Non-lipidated variants of neisseria meningitidis orf2086 antigens

ABSTRACT

The present invention relates to compositions including an isolated non-pyruvylated non-lipidated ORF2086 polypeptide, and methods thereof. In an exemplary embodiment, the compositions described herein are immunogenic. The present invention further relates to compositions that elicit a bactericidal immune response in a mammal against an ORF2086 subfamily B polypeptide, from serogroup B Neisseria meningitides, and methods related thereto.

This is a continuation of U.S. patent application Ser. No. 15/587,574,filed May 5, 2017, which is a continuation of U.S. patent applicationSer. No. 13/295,030, filed on Nov. 11, 2011, which is a continuation ofInternational Application Number PCT/IB2011/05393, filed Sep. 8, 2011,which claims the benefit of US Provisional Patent Application Number61/381,837, filed Sep. 10, 2010. The entire contents of theaforementioned applications are herein incorporated by reference intheir entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingfor the 10 application, which is disclosed on a computer-readable ASCIItext file, created on Sep. 8, 2011, named Untitled_ST25.txt, and is73965 bytes in size.

FIELD OF THE INVENTION

The present invention relates to non-lipidated variants of Neisseriameningitidis ORF2086 antigens in immunogenic compositions as describedherein. The present invention also relates to methods of preserving theconformation of non-lipidated variants of Neisseria meningitidis ORF2086antigens. The present invention further includes compositions andmethods relating to improved expression of non-lipidated N. meningitidisORF2086 antigens, as compared to the corresponding wild-type antigen.

BACKGROUND OF THE INVENTION

rLP2086 is a recombinant 28-kDa lipoprotein that induces cross-reactivebacterial antibodies against a number of Neisseria meningitidis strains,including Neisseria meningitidis serotype B (MnB) strains, or moreprecisely, serogroup B (MnB) strains. Based on deduced amino acidsequence homology, two different subfamilies of rLP2086 were identified,A and B. These two subfamilies were used in the formulation of theMnB-rLP2086 vaccine samples containing 20, 60,120, and 200 μg/mL each in10 mM Histidine (pH 6.0), 150 mM NaCl, and 0.5 mg/mL aluminum withvarying levels of Polysorbate 80 (PS-80). Native LP2086 is alipoprotein. Fletcher et al. Infection & Immunity. vol. 72(4):2088-2100(2004) demonstrated that rLP2086 with an amino terminal lipid was moreimmunogenic than non-lipidated versions of the same protein in mice.Additional preclinical and clinical studies have demonstrated that thecombination of these two lipidated proteins can provide broad coverageacross the fHBP family . Meningococcal meningitis is a devastatingdisease that can kill children and young adults within hours despite theavailability of antibiotics. There remains a need for suitable serogroupB meningococcal immunogenic compositions.

SUMMARY OF THE INVENTION

To meet these and other needs for a meningococcal vaccine, additionalcompositions have been evaluated to provide coverage for usingnon-lipidated variants of N. meningitidis ORF2086 polypeptides. A firstaspect of the present invention provides an immunogenic compositioncomprising a non-lipidated ORF2086 protein, wherein the ORF2086 proteinis a B44, a B02, a B03, a B22, a B24, a B09, an A05, an A04, an A12, oran A22 variant. In some embodiments, the ORF2086 protein is a B44, aB22, a B09, an A05, an A12, or an A22 variant.

Another aspect of the present invention provides an immunogeniccomposition comprising a non-lipidated ORF2086 protein Subfamily Bvariant (P2086 Subfamily B polypeptide). In some embodiments, the P2086Subfamily B polypeptide is a B44, a B02, a B03, a B22, a B24, or a B09variant. In some embodiments, the immunogenic composition furthercomprises a non-lipidated ORF2086 protein Subfamily A variant (P2086Subfamily A polypeptide). In some embodiments, the P2086 Subfamily Apolypeptide is an A05, an A04, an A12, or an A22 variant.

In some embodiments, the immunogenic composition further comprises anadjuvant. In some embodiments, the adjuvant is an aluminum adjuvant, asaponin, a CpG nucleotide sequence or any combination thereof. In someembodiments, the aluminum adjuvant is AlPO₄, Al(OH)₃, Al₂(SO₄)₃, oralum. In some embodiments the concentration of aluminum in theimmunogenic composition is between 0.125 μg/ml and 0.5 μg/ml. In someembodiments the concentration of aluminum in the immunogenic compositionis 0.25 μg/ml. In a preferred embodiment, the concentration of aluminumin the immunogenic composition is between 0.125 mg/ml and 0.5 mg/ml. Insome preferred embodiments the concentration of aluminum in theimmunogenic composition is 0.25 mg/ml.

In some embodiments, the saponin concentration in the immunogeniccomposition is between 1 μg/ml and 250 μg/ml. In some embodiments, thesaponin concentration in the immunogenic composition is between 10 μg/mland 100 μg/ml. In some embodiments, the saponin concentration in theimmunogenic composition is 10 μg/ml. In some embodiments, the saponinconcentration in the immunogenic composition is 100 μg/ml. In someembodiments, the saponin is QS-21 Stimulon® (Agenus, Lexington, Mass.)or ISCOMATRIX® (CSL Limited, Parkville, Australia).

In some embodiments, the immunogenic composition confers the ability toraise an immunogenic response to Neisseria meningitidis afteradministration of multiple doses of the immunogenic composition to asubject. In some embodiments, the immunogenic response is conferredafter administration of two doses to the subject. In some embodiments,the immunogenic response is conferred after administration of threedoses to the subject.

Another aspect of the invention provides a composition conferringincreased immunogenicity of a non-lipidated P2086 antigen, wherein thecomposition comprises a saponin and at least one non-lipidated P2086antigen. In some embodiments, the saponin concentration in theimmunogenic composition is between 1 μg/ml and 250 μg/ml. In someembodiments, the saponin concentration in the immunogenic composition isbetween 10 μg/ml and 100 μg/ml. In some embodiments, the saponinconcentration in the immunogenic composition is 10 μg/ml. In someembodiments, the saponin concentration in the immunogenic composition is100 μg/ml. In some embodiments, the saponin is QS-21 or ISCOMATRIX.

In some embodiments, the composition further comprises aluminum. In someembodiments, the aluminum is present as AlPO₄, Al(OH)₃, Al₂(SO₄)₃, oralum. In some embodiments the concentration of aluminum in thecomposition is between 0.125 μg/ml and 0.5 μg/ml. In some embodimentsthe concentration of aluminum in the composition is 0.25 μg/ml. In apreferred embodiment, the concentration of aluminum in the compositionis between 0.125 mg/ml and 0.5 mg/ml. In some preferred embodiments theconcentration of aluminum in the composition is 0.25 mg/ml.

In some embodiments, the immunogenic composition confers the ability toraise an immunogenic response to Neisseria meningitidis afteradministration of multiple doses of the immunogenic composition to asubject. In some embodiments, the immunogenic response is conferredafter administration of two doses to the subject. In some embodiments,the immunogenic response is conferred after administration of threedoses to the subject.

In some embodiments, the non-lipidated P2086 antigen is a P2086Subfamily B polypeptide. In some embodiments, the P2086 Subfamily Bpolypeptide is a B44, a B02, a B03, a B22, a B24 or a B09 variant. Insome embodiments, the non-lipidated P2086 antigen is a P2086 Subfamily Apolypeptide. In some embodiment, the P2086 Subfamily A polypeptide is anA05, an A04, an A12, or an A22 variant.

In some embodiments, the composition comprises at least twonon-lipidated P2086 antigens, wherein the two non-lipidated P2086antigens are at least one non-lipidated P2086 Subfamily A polypeptideand at least one non-lipidated P2086 Subfamily B polypeptide. In someembodiments, the non-lipidated P2086 Subfamily A polypeptide is an A05variant and the non-lipidated P2086 Subfamily B polypeptide is a B44variant. In some embodiments, the non-lipidated P2086 Subfamily Apolypeptide is an A05 variant and the non-lipidated P2086 Subfamily Bpolypeptide is a B22 variant. In some embodiments, the non-lipidatedP2086 Subfamily A polypeptide is an A05 variant and the non-lipidatedP2086 Subfamily B polypeptide is a B09 variant.

Another aspect of the invention provides a method for conferringimmunity to a subject against a Neisseria meningitidis bacteria, whereinthe method comprises the step of administering to the subject animmunogenic composition comprising a non-lipidated P2086 Subfamily Bpolypeptide. In some embodiments, the P2086 Subfamily B polypeptide is aB44, a B02, a B03, a B22, a B24 or a B09 variant. In some embodiments,the immunogenic composition further comprises a P2086 Subfamily Apolypeptide. In some embodiments, the P2086 Subfamily A polypeptide isan A05, an A04, an A12, or an A22 variant.

In some embodiments, the immunogenic composition further comprises anadjuvant. In some embodiments, the adjuvant is an aluminum adjuvant, asaponin, a CpG nucleotide sequence or any combination thereof. In someembodiments, the aluminum adjuvant is AlPO₄, Al(OH)₃, Al₂(SO₄)₃, oralum. In some embodiments, the concentration of aluminum in theimmunogenic composition is between 0.125 μg/ml and 0.5 μg/ml. In someembodiments, the concentration of aluminum in the immunogeniccomposition is 0.25 μg/ml. In a preferred embodiment, the concentrationof aluminum in the immunogenic composition is between 0.125 mg/ml and0.5 mg/ml. In some embodiments, the concentration of aluminum in theimmunogenic composition is 0.25 mg/ml.

In some embodiments, the saponin concentration in the immunogeniccomposition is between 1 μg/ml and 250 μg/ml. In some embodiments, thesaponin concentration in the immunogenic composition is between 10 μg/mland 100 μg/ml. In some embodiments, the saponin concentration in theimmunogenic composition is 10 μg/ml. In some embodiments, the saponinconcentration in the immunogenic composition is 100 μg/ml. In someembodiments, the saponin is QS-21 or ISCOMATRIX.

In some embodiments, the immunogenic composition is administered to thesubject in multiple doses over a dosing schedule. In some embodiments,the immunogenic composition is administered to the subject in two dosesover a dosing schedule. In some embodiments, the immunogenic compositionis administered to the subject in three doses over a dosing schedule.

Another aspect of the invention provides a method of producing anon-lipidated P2086 variant comprising the steps of (a) cloning anORF2086 variant nucleic acid into an expression vector to generate anORF2086 expression vector; (b) transforming bacteria with the OFR2086expression vector; (c) inducing expression of the P2086 variant from theORF2086 expression vector; and (d) isolating the expressed P2086 variantprotein; wherein the ORF2086 expression vector does not comprise alipidation control sequence. In some embodiments, the bacteria is E.coli. In some embodiments, expression is induced by addition of IPTG.

In some embodiments, the codon encoding the N-terminal Cys of the P2086variant is deleted. In some embodiments, the codon encoding theN-terminal Cys of the P2086 variant is mutated to generate an Ala, Glyor Val codon. In some embodiments, the P2086 variant is an A05, B01, orB44 variant. In some embodiments, the P2086 variant is a B09 variant.

In some embodiments, the N-terminal tail is mutated to add Ser and Glyresidues to extend the Gly/Ser stalk immediately downstream of theN-terminal Cys. In some embodiments, the total number of Gly and Serresidues in the Gly/Ser stalk is at least 7, at least 8, at least 9, atleast 10, at least 11, or at least 12.

In some embodiments, the codons of the N-terminal tail of the P2086variant are optimized by point mutagenesis. In some embodiments, thecodons of the N-terminal tail of the ORF2086 variant are optimized bypoint mutagenesis such that the codon encoding the fifth amino acid ofthe ORF2086 variant is 100% identical to nucleotides 13-15 of SEQ ID NO:8 and the codon encoding the thirteenth amino acid of the ORF2086variant is 100% identical to nucleotides 37-39 of SEQ ID NO: 8. In someembodiments, the N-terminal tail of the non-lipidated ORF2086 variant isoptimized such that the 5′ 45 nucleic acids are 100% identical tonucleic acids 1-45 of SEQ ID NO: 8. In some embodiments, the N-terminaltail of the non-lipidated ORF2086 variant is optimized such that the 5′42 nucleic acids are 100% identical to nucleic acids 4-45 of SEQ ID NO:8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086variant is optimized such that the 5′ 39 nucleic acids are 100%identical to nucleic acids 4-42 of SEQ ID NO: 8. In some embodiments,the N-terminal tail of the non-lipidated P2086 variant comprises atleast one amino acid substitution compared to amino acids 1-15 of SEQ IDNO: 18. In some embodiments, the N-terminal tail of the non-lipidatedP2086 variant comprises two amino acid substitutions compared to aminoacids 1-15 of SEQ ID NO: 18. In some embodiments, the N-terminal tail ofthe non-lipidated P2086 variant comprises at least one amino acidsubstitution compared to amino acids 2-15 of SEQ ID NO: 18. In someembodiments, the N-terminal tail of the non-lipidated P2086 variantcomprises two amino acid substitutions compared to amino acids 2-15 ofSEQ ID NO: 18. In some embodiments, the amino acid substitutions areconservative amino acid substitutions.

In one embodiment, the present invention relates to stable formulationsof Neisseria meningitis ORF2086 Subfamily B Antigens in immunogeniccompositions.

The present invention also relates to methods of preserving theconformation of Neisseria meningitis ORF2086 Antigens and methods fordetermining the potency of Neisseria meningitis rLP2086 antigens.

In one aspect, the invention relates to a composition that includes anisolated non-pyruvylated non-lipidated ORF2086 polypeptide. In oneembodiment, the composition is immunogenic. In another embodiment, thepolypeptide includes a deletion of an N-terminal Cys compared to thecorresponding wild-type non-lipidated ORF2086 polypeptide. In oneembodiment, the polypeptide includes the amino acid sequence selectedfrom the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16 SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 isdeleted. In another embodiment, the polypeptide includes the amino acidsequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO:49, SEQ ID NO: 50, and SEQ ID NO: 55.

In yet another embodiment, the polypeptide is encoded by a nucleotidesequence that is operatively linked to an expression system, whereinsaid expression system is capable of being expressed in a bacterialcell. In one embodiment, the expression system is a plasmid expressionsystem. In one embodiment, the bacterial cell is an E. coli cell. Inanother embodiment, the nucleotide sequence is linked to a regulatorysequence that controls expression of said nucleotide sequence.

In another aspect, the invention relates to a composition that includesa non-pyruvylated non-lipidated ORF2086 polypeptide obtainable by aprocess. The process includes expressing a nucleotide sequence encodinga polypeptide having the amino acid sequence selected from the groupconsisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16 SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO: 20,and SEQ ID NO: 21, wherein the cysteine at position 1 is deleted,wherein the nucleotide sequence is operatively linked to an expressionsystem that is capable of being expressed in a bacterial cell. In oneembodiment, the bacterial cell is E. coli.

In one aspect, the invention relates to a composition that includes anisolated polypeptide, which includes the amino acid sequence set forthin SEQ ID NO: 49, and an isolated polypeptide, which includes the aminoacid sequence set forth in SEQ ID NO: 44. In one embodiment, thecompositions described herein are immunogenic. In another embodiment,the compositions described herein further include an ORF2086 subfamily Apolypeptide from serogroup B N. meningitidis. In another embodiment,compositions described herein elicit a bactericidal immune response in amammal against an ORF2086 subfamily B polypeptide from serogroup B N.meningitidis.

In one aspect, the invention relates to an isolated polypeptide thatincludes the amino acid sequence set forth in SEQ ID NO: 49. In anotheraspect, the invention relates to an isolated nucleotide sequence thatincludes SEQ ID NO: 46. In one aspect, the invention relates to anisolated nucleotide sequence that includes SEQ ID NO: 47. In one aspect,the invention relates to an isolated nucleotide sequence that includesSEQ ID NO: 48. In one aspect, the invention relates to an isolatedpolypeptide that includes the amino acid sequence set forth in SEQ IDNO: 50. In one aspect, the invention relates to an isolated nucleotidesequence that includes SEQ ID NO: 45. In one aspect, the inventionrelates to an isolated polypeptide that includes the amino acid sequenceset forth in SEQ ID NO: 44.

In one aspect, the invention relates to a plasmid that includes anucleotide sequence selected from the group consisting of SEQ ID NO: 46,SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 45, wherein the plasmid iscapable of being expressed in a bacterial cell. In one embodiment, thebacterial cell is E. coll.

In one aspect, the invention relates to a method of elicitingbactericidal antibodies specific to an ORF2086 subfamily B serogroup BN. meningitidis in a mammal. The method includes administering to themammal an effective amount of an isolated polypeptide that includes theamino acid sequence selected from the group consisting of SEQ ID NO: 44and SEQ ID NO: 49, or a combination thereof.

In one aspect, the invention relates to a method for producing apolypeptide. The method includes expressing in a bacterial cell apolypeptide, which includes a sequence having greater than 90% identityto SEQ ID NO:21, said sequence including at least one domain selectedfrom the group consisting of amino acids 13-18 of SEQ ID NO: 21, aminoacids 21-34 of SEQ ID NO: 21, and amino acids 70-80 of SEQ ID NO: 21, ora combination thereof, wherein the sequence lacks an N-terminalcysteine. The method further includes purifying the polypeptide. In oneembodiment, the sequence further includes at least one domain selectedfrom the group consisting of amino acids 96-116 of SEQ ID NO: 21, aminoacids 158-170 of SEQ ID NO: 21, amino acids 172-185 of SEQ ID NO: 21,amino acids 187-199 of SEQ ID NO: 21, amino acids 213-224 of SEQ ID NO:21, amino acids 226-237 of SEQ ID NO: 21, amino acids 239-248 of SEQ IDNO: 21, or a combination thereof. In one embodiment, the bacterial cellis E. coli.

In one aspect, the invention relates to an isolated polypeptide producedby a process that includes the method described herein. In anotheraspect, the invention relates to an immunogenic composition produced bya process that includes the method described herein.

In one aspect, the invention relates to an immunogenic composition thatincludes an ORF2086 subfamily B polypeptide from serogroup B N.meningitidis, wherein the polypeptide is a non-pyruvylated non-lipidatedB44. In one embodiment, the composition further includes a secondORF2086 subfamily B polypeptide from serogroup B N. meningitidis,wherein the second polypeptide is a non-pyruvylated non-lipidated B09.In one embodiment, the composition includes no more than 3 ORF2086subfamily B polypeptides. In another embodiment, the compositionincludes no more than 2 ORF2086 subfamily B polypeptides. In oneembodiment, the composition further includes a ORF2086 subfamily Apolypeptide. In another embodiment, the composition includes an A05subfamily A polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F: P2086 Variant Nucleic Acid Sequences.

FIGS. 2A to 2C: P2086 Variant Amino Acid Sequences. The Gly/Ser stalk inthe N-terminal tail of each variant is underlined.

FIG. 3: Structure of the ORF2086 Protein

FIG. 4: Removal of N-terminal Cys Results in Loss of Expression in E.coli.

FIG. 5: Effect of Gly/Ser Stalk Length on Non-lipidated ORF2086 VariantExpression. The sequence associated with the protein variant labeled B01is set forth in SEQ ID NO: 35. The sequence associated with the proteinvariant labeled B44 is set forth in SEQ ID NO: 36. The sequenceassociated with the protein variant labeled A05 is set forth in SEQ IDNO: 37. The sequence associated with the protein variant labeled A22 isset forth in SEQ ID NO: 38. The sequence associated with the proteinvariant labeled B22 is set forth in SEQ ID NO: 39. The sequenceassociated with the protein variant labeled A19 is set forth in SEQ IDNO: 40.

FIG. 6: High Levels of Non-lipidated B09 Expression Despite A ShortGly/Ser Stalk. The left two lanes demonstrated expression of theN-terminal Cys-deleted B09 variant before and after induction. The thirdand fourth lanes demonstrate expression of the N-terminal Cys positiveB09 variant before and after induction. The right most lane is amolecular weight standard. The amino acid sequence shown under the imageis set forth in SEQ ID NO: 41. The nucleotide sequence representative ofthe N-terminal Cys-deleted A22 variant, referred to as “A22_001” in thefigure, is set forth in SEQ ID NO: 42, which is shown under SEQ ID NO:41 in the figure. The nucleotide sequence representative of theN-terminal Cys-deleted B22 variant, referred to as “B22_001” in thefigure, is set forth in SEQ ID NO: 52. The nucleotide sequencerepresentative of the N-terminal Cys-deleted B09 variant, referred to as“B09_004” in the figure, is set forth in SEQ ID NO: 53.

FIG. 7: Codon Optimization Increases Expression of Non-lipidated B22 andA22 Variants. The left panel demonstrates expression of the N-terminalCys-deleted B22 variant before (lanes 1 and 3) and after (lanes 2 and 4)IPTG induction. The right panel demonstrates expression of theN-terminal Cys-deleted A22 variant before (lane 7) and after (lane 8)IPTG induction. Lanes 5 and 6 are molecular weight standards.

FIGS. 8A to 8H: P2086 Variant Nucleic and Amino Acid Sequences

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant A04 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 2 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant A05 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 3 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant A12 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 4 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant A12-2 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 5 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant A22 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 6 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B02 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 7 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B03 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 8 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B09 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 9 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B22 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 10 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B24 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 11 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B44 gene, which includes a codon encoding anN-terminal Cys.

SEQ ID NO: 12 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant A04, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 13 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant A05, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 14 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant A12, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 15 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant A22, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 16 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B02, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 17 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B03, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 18 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B09, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 19 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B22, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 20 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B24, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 21 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B44, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 22 sets forth a DNA sequence for a forward primer, shown inExample 2.

SEQ ID NO: 23 sets forth a DNA sequence for a reverse primer, shown inExample 2.

SEQ ID NO: 24 sets forth a DNA sequence for a forward primer, shown inExample 2, Table 1.

SEQ ID NO: 25 sets forth a DNA sequence for a reverse primer, shown inExample 2, Table 1.

SEQ ID NO: 26 sets forth a DNA sequence for a forward primer, shown inExample 2, Table 1.

SEQ ID NO: 27 sets forth a DNA sequence for a reverse primer, shown inExample 2, Table 1.

SEQ ID NO: 28 sets forth a DNA sequence for a Gly/Ser stalk, shown inExample 4.

SEQ ID NO: 29 sets forth the amino acid sequence for a Gly/Ser stalk,shown in Example 4, which is encoded by, for example SEQ ID NO: 28.

SEQ ID NO: 30 sets forth a DNA sequence for a Gly/Ser stalk, shown inExample 4.

SEQ ID NO: 31 sets forth the amino acid sequence a Gly/Ser stalk, shownin Example 4, which is encoded by, for example SEQ ID NO: 30.

SEQ ID NO: 32 sets forth a DNA sequence for a Gly/Ser stalk, shown inExample 4.

SEQ ID NO: 33 sets forth the amino acid sequence for a Gly/Ser stalk,which is encoded by, for example, SEQ ID NO: 32 and SEQ ID NO: 34.

SEQ ID NO: 34 sets forth a DNA sequence for a Gly/Ser stalk, shown inExample 4.

SEQ ID NO: 35 sets forth the amino acid sequence for the N-terminus ofN. meningitidis, serogroup B, 2086 variant B01, shown in FIG. 5.

SEQ ID NO: 36 sets forth the amino acid sequence for the N-terminus ofN. meningitidis, serogroup B, 2086 variant B44, shown in FIG. 5.

SEQ ID NO: 37 sets forth the amino acid sequence for the N-terminus ofN. meningitidis, serogroup B, 2086 variant A05, shown in FIG. 5.

SEQ ID NO: 38 sets forth the amino acid sequence for the N-terminus ofN. meningitidis, serogroup B, 2086 variant A22, shown in FIG. 5.

SEQ ID NO: 39 sets forth the amino acid sequence for the N-terminus ofN. meningitidis, serogroup B, 2086 variant B22, shown in FIG. 5.

SEQ ID NO: 40 sets forth the amino acid sequence for the N-terminus ofN. meningitidis, serogroup B, 2086 variant A19, shown in FIG. 5.

SEQ ID NO: 41 sets forth the amino acid sequence for the N-terminus of aN. meningitidis, serogroup B, 2086 variant, shown in FIG. 6.

SEQ ID NO: 42 sets forth a DNA sequence for the N-terminus of N.meningitidis, serogroup B, 2086 variant A22, shown in FIG. 6.

SEQ ID NO: 43 sets forth a codon-optimized DNA sequence for the N.meningitidis, serogroup B, 2086 variant B44 gene, wherein the codonencoding an N-terminal cysteine is deleted, as compared to SEQ ID NO:11. Plasmid pDK087 includes SEQ ID NO: 43.

SEQ ID NO: 44 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B44. SEQ ID NO: 44 is identicalto SEQ ID NO: 21 wherein the N-terminal cysteine at position 1 of SEQ IDNO: 21 is deleted. SEQ ID 44 is encoded by, for example, SEQ ID NO: 43.

SEQ ID NO: 45 sets forth a codon-optimized DNA sequence for the N.meningitidis, serogroup B, 2086 variant B09 gene, wherein the codonencoding an N-terminal cysteine is deleted, and wherein the sequenceincludes codons encoding an additional Gly/Ser region, as compared toSEQ ID NO: 8. Plasmid pEB063 includes SEQ ID NO: 45.

SEQ ID NO: 46 sets forth a codon-optimized DNA sequence for the N.meningitidis, serogroup B, 2086 variant B09 gene, wherein the codonencoding an N-terminal cysteine is deleted, as compared to SEQ ID NO: 8.Plasmid pEB064 includes SEQ ID NO: 46.

SEQ ID NO: 47 sets forth a codon-optimized DNA sequence for the N.meningitidis, serogroup B, 2086 variant B09 gene, wherein the codonencoding an N-terminal cysteine is deleted, as compared to SEQ ID NO: 8.Plasmid pEB 065 includes SEQ ID NO: 47.

SEQ ID NO: 48 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B09 gene, wherein the codon encoding anN-terminal cysteine is deleted, as compared to SEQ ID NO: 8. PlasmidpLA134 includes SEQ ID NO: 48.

SEQ ID NO: 49 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B09. SEQ ID NO: 49 is identicalto SEQ ID NO: 18 wherein the N-terminal cysteine at position 1 of SEQ IDNO: 18 is deleted. SEQ ID 49 is encoded by, for example, a DNA sequenceselected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, andSEQ ID NO: 48.

SEQ ID NO: 50 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B09, wherein the codon encodingan N-terminal cysteine is deleted and wherein the sequence includescodons encoding an additional Gly/Ser region, as compared to SEQ ID NO:18. SEQ ID NO: 50 is encoded by, for example, SEQ ID NO: 45.

SEQ ID NO: 51 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B44 gene, wherein the codon encoding anN-terminal cysteine is deleted, as compared to SEQ ID NO: 11. PlasmidpLN056 includes SEQ ID NO: 51.

SEQ ID NO: 52 sets forth a DNA sequence for the N-terminus of N.meningitidis, serogroup B, 2086 variant B22, shown in FIG. 6.

SEQ ID NO: 53 sets forth a DNA sequence for the N-terminus of N.meningitidis, serogroup B, 2086 variant B09, shown in FIG. 6.

SEQ ID NO: 54 sets forth a DNA sequence for a N. meningitidis, serogroupB, 2086 variant A05 gene, wherein the codon encoding an N-terminalcysteine is deleted, as compared to SEQ ID NO: 2.

SEQ ID NO: 55 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A05. SEQ ID NO: 55 is identicalto SEQ ID NO: 13 wherein the N-terminal cysteine at position 1 of SEQ IDNO: 13 is deleted. SEQ ID NO: 55 is encoded by, for example, SEQ ID NO:54.

SEQ ID NO: 56 sets forth the amino acid sequence of a serine-glycinerepeat sequence, shown in Example 7.

SEQ ID NO: 57 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B01. SEQ ID NO: 57 is identicalto SEQ ID NO: 58 wherein the N-terminal cysteine at position 1 of SEQ IDNO: 58 is deleted.

SEQ ID NO: 58 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B01, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 59 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B15, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 60 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B16, which includes anN-terminal Cys at amino acid position 1.

SEQ ID NO: 61 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant B22,in which the codon for the N-terminal Cysat amino acid position 1 of SEQ ID NO: 19 is replaced with a codon for aGlycine.

SEQ ID NO: 62 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant B22, in which the N-terminal Cysat amino acid position 1 of SEQ ID NO: 19 is replaced with a Glycine.

SEQ ID NO: 63 sets forth a DNA sequence for the N. meningitidis,serogroup B, 2086 variant A22,in which the codon for the N-terminal Cysat amino acid position 1 of SEQ ID NO: 15 is replaced with a codon foraGlycine.

SEQ ID NO: 64 sets forth the amino acid sequence for the N.meningitidis, serogroup B, 2086 variant A22, in which the N-terminal Cysat amino acid position 1 of SEQ ID NO: 15 is replaced with a Glycine.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting. Allpublications, patents and other documents mentioned herein areincorporated by reference in their entirety.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. patent law; e.g., they canmean “includes”, “included”, “including” and the like. Such terms referto the inclusion of a particular ingredients or set of ingredientswithout excluding any other ingredients. Terms such as “consistingessentially of” and “consists essentially of” have the meaningattributed to them in U.S. patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the invention, and they exclude ingredients orsteps of the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, non-obvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. patent law; namely, that these terms are close-ended. Accordingly,these terms refer to the inclusion of a particular ingredient or set ofingredients and the exclusion of all other ingredients.

Definitions

As used herein, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, e.g.,references to “the method” includes one or more methods, and/or steps ofthe type described herein and/or which will become apparent to one ofordinary skill in the art upon reading this disclosure and so forth.

As used herein, the plural forms include singular references unless thecontext clearly dictates otherwise. Thus, e.g., references to “themethods” includes one or more methods, and/or steps of the typedescribed herein and/or which will become apparent to one of ordinaryskill in the art upon reading this disclosure and so forth.

As used herein, “about” means within a statistically meaningful range ofa value such as a stated concentration range, time frame, molecularweight, temperature or pH. Such a range can be within an order ofmagnitude, typically within 20%, more typically still within 10%, andeven more typically within 5% of a given value or range. The allowablevariation encompassed by the term “about” will depend upon theparticular system under study, and can be readily appreciated by one ofordinary skill in the art. Whenever a range is recited within thisapplication, every whole number integer within the range is alsocontemplated as an embodiment of the invention.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen as further described and exemplifiedherein. Non-limiting examples of adjuvants that can be used in thevaccine of the present invention include the RIBI adjuvant system (RibiInc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxidegel, oil-in-water emulsions, water-in-oil emulsions such as, e.g.,Freund's complete and incomplete adjuvants, Block copolymer (CytRx,Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M(Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A orother saponin fraction, monophosphoryl lipid A, and Avridine lipid-amineadjuvant.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, unlessotherwise indicated by context, the term is intended to encompass notonly intact polyclonal or monoclonal antibodies, but also engineeredantibodies (e.g., chimeric, humanized and/or derivatized to altereffector functions, stability and other biological activities) andfragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv)and domain antibodies, including shark and camelid antibodies), andfusion proteins comprising an antibody portion, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies so long as theyexhibit the desired biological activity) and antibody fragments asdescribed herein, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site. Anantibody includes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 inhumans. The heavy-chain constant domains that correspond to thedifferent classes of immunoglobulins are called alpha, delta, epsilon,gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody.

The term “antigen” generally refers to a biological molecule, usually aprotein, peptide, polysaccharide, lipid or conjugate which contains atleast one epitope to which a cognate antibody can selectively bind; orin some instances to an immunogenic substance that can stimulate theproduction of antibodies or T-cell responses, or both, in an animal,including compositions that are injected or absorbed into an animal. Theimmune response may be generated to the whole molecule, or to one ormore various portions of the molecule (e.g., an epitope or hapten). Theterm may be used to refer to an individual molecule or to a homogeneousor heterogeneous population of antigenic molecules. An antigen isrecognized by antibodies, T-cell receptors or other elements of specifichumoral and/or cellular immunity. The term “antigen” includes allrelated antigenic epitopes. Epitopes of a given antigen can beidentified using any number of epitope mapping techniques, well known inthe art. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.For example, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes may be identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Furthermore, for purposes of the present invention, an“antigen” may also be used to refer to a protein that includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature, but they may be non-conservative), to the nativesequence, so long as the protein maintains the ability to elicit animmunological response. These modifications may be deliberate, asthrough site-directed mutagenesis, or through particular syntheticprocedures, or through a genetic engineering approach, or may beaccidental, such as through mutations of hosts, which produce theantigens. Furthermore, the antigen can be derived, obtained, or isolatedfrom a microbe, e.g. a bacterium, or can be a whole organism. Similarly,an oligonucleotide or polynucleotide, which expresses an antigen, suchas in nucleic acid immunization applications, is also included in thedefinition. Synthetic antigens are also included, for example,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens (Bergmann et al. (1993) Eur. J. lmmunol. 23:2777 2781;Bergmann et al. (1996) J. lmmunol. 157:3242 3249; Suhrbier, A. (1997)lmmunol. and Cell Biol. 75:402 408; Gardner et al. (1998) 12th WorldAIDS Conference, Geneva, Switzerland, Jun. 28-Jul. 3, 1998).

The term “conservative” amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, tryptophan, and methionine;polar/neutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine, and glutamine; positively charged (basic) aminoacids include arginine, lysine, and histidine; and negatively charged(acidic) amino acids include aspartic acid and glutamic acid. In someembodiments, the conservative amino acid changes alter the primarysequence of the ORF2086 polypeptides, but do not alter the function ofthe molecule. When generating these mutants, the hydropathic index ofamino acids can be considered. The importance of the hydropathic aminoacid index in conferring interactive biologic function on a polypeptideis generally understood in the art (Kyte & Doolittle, 1982, J. Mol.Biol., 157(1):105-32). It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino acidresidue determines the secondary and tertiary structure of the resultantpolypeptide, which in turn defines the interaction of the polypeptidewith other molecules, such as enzymes, substrates, receptors,antibodies, antigens, and the like. It is known in the art that an aminoacid can be substituted by another amino acid having a similarhydropathic index and still obtain a functionally equivalentpolypeptide. In such changes, the substitution of amino acids whosehydropathic indices are within +/−2 is preferred, those within +/−1 areparticularly preferred, and those within +/−0.5 are even moreparticularly preferred.

Conservative amino acids substitutions or insertions can also be made onthe basis of hydrophilicity. As described in U.S. Pat. No. 4,554,101,which is hereby incorporated by reference the greatest local averagehydrophilicity of a polypeptide, as governed by the hydrophilicity ofits adjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the polypeptide. U.S.Pat. No. 4,554,101reciates that the following hydrophilicity values havebeen assigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred; those within ±1 are particularly preferred; and thosewithin ±0.5 are even more particularly preferred. Exemplarysubstitutions which take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include,without limitation: arginine and lysine; glutamate and aspartate; serineand threonine; glutamine and asparagine; and valine, leucine andisoleucine.

The term “effective immunogenic amount” as used herein refers to anamount of a polypeptide or composition comprising a polypeptide which iseffective in eliciting an immune response in a vertebrate host. Forexample, an effective immunogenic amount of a rLP2086 protein of thisinvention is an amount that is effective in eliciting an immune responsein a vertebrate host. The particular “effective immunogenic dosage oramount” will depend upon the age, weight and medical condition of thehost, as well as on the method of administration. Suitable doses arereadily determined by persons skilled in the art.

The term “Gly/Ser stalk” as used herein refers to the series of Gly andSer residues immediately downstream of the N-terminal Cys residue of aprotein encoded by ORF2086. There can be between 5 and 12 Gly and Serresidues in the Gly/Ser stalk. Accordingly, the Gly/Ser stalk consistsof amino acids 2 to between 7 and 13 of the protein encoded by ORF2086.Preferably, the Gly/Ser stalk consists of amino acids 2 and up tobetween 7 and 13 of the protein encoded by ORF2086. The Gly/Ser stalksof the P2086 variants of the present invention are represented by theunderlined sequences in FIG. 2 (SEQ ID NO: 12-21). As shown herein, thelength of the Gly/Ser stalk can affect the stability or expression levelof a non-lipidated P2086 variant. In an exemplary embodiment, effectsfrom affecting the length of the Gly/Ser stalk are compared to thosefrom the corresponding wild-type variant.

The term “immunogenic” refers to the ability of an antigen or a vaccineto elicit an immune response, either humoral or cell-mediated, or both.

An “immunogenic amount”, or an “immunologically effective amount” or“dose”, each of which is used interchangeably herein, generally refersto the amount of antigen or immunogenic composition sufficient to elicitan immune response, either a cellular (T cell) or humoral (B cell orantibody) response, or both, as measured by standard assays known to oneskilled in the art.

The term “immunogenic composition” relates to any pharmaceuticalcomposition containing an antigen, e.g. a microorganism, or a componentthereof, which composition can be used to elicit an immune response in asubject. The immunogenic compositions of the present invention can beused to treat a human susceptible to N. meningidis infection, by meansof administering the immunogenic compositions via a systemic transdermalor mucosal route. These administrations can include injection via theintramuscular (i.m.), intraperitoneal (i.p.), intradermal (i.d.) orsubcutaneous routes; application by a patch or other transdermaldelivery device; or via mucosal administration to the oral/alimentary,respiratory or genitourinary tracts. In one embodiment, the immunogeniccomposition may be used in the manufacture of a vaccine or in theelicitation of a polyclonal or monoclonal antibodies that could be usedto passively protect or treat a subject.

Optimal amounts of components for a particular immunogenic compositioncan be ascertained by standard studies involving observation ofappropriate immune responses in subjects. Following an initialvaccination, subjects can receive one or several booster immunizationsadequately spaced.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturally occurringor from it's host organism if it is a recombinant entity, or taken fromone environment to a different environment). For example, an “isolated”protein or peptide is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized, or otherwise present in amixture as part of a chemical reaction. In the present invention, theproteins may be isolated from the bacterial cell or from cellulardebris, so that they are provided in a form useful in the manufacture ofan immunogenic composition. The term “isolated” or “isolating” mayinclude purifying, or purification, including for example, the methodsof purification of the proteins, as described herein. The language“substantially free of cellular material” includes preparations of apolypeptide or protein in which the polypeptide or protein is separatedfrom cellular components of the cells from which it is isolated orrecombinantly produced. Thus, a protein or peptide that is substantiallyfree of cellular material includes preparations of the capsulepolysaccharide, protein or peptide having less than about 30%, 20%, 10%,5%, 2.5%, or 1%, (by dry weight) of contaminating protein orpolysaccharide or other cellular material. When the polypeptide/proteinis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When polypeptide orprotein is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein or polysaccharide. Accordingly,such preparations of the polypeptide or protein have less than about30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compoundsother than polypeptide/protein or polysaccharide fragment of interest.

The term “N-terminal tail” as used herein refers to the N-terminalportion of a protein encoded by ORF2086, which attaches the protein tothe cell membrane. An N-terminal tail is shown at the bottom of the sideview structure in FIG. 3. An N-terminal tail typically comprises theN-terminal 16 amino acids of the protein encoded by ORF2086. In someembodiments, the N-terminal tail is amino acids 1-16 of any one of SEQID NOs: 12-21.The term “ORF2086” as used herein refers to Open ReadingFrame 2086 from a Neisseria species bacteria. Neisseria ORF2086, theproteins encoded therefrom, fragments of those proteins, and immunogeniccompositions comprising those proteins are known in the art and aredescribed, e.g., in WO2003/063766, and in U.S. Patent ApplicationPublication Nos. US 20060257413 and US 20090202593, each of which ishereby incorporated by reference in its entirety.

The term “P2086” generally refers to the protein encoded by ORF2086. The“P” before “2086” is an abbreviation for “protein.” The P2086 proteinsof the invention may be lipidated or non-lipidated. “LP2086” and “P2086”typically refer to lipidated and non-lipidated forms of a 2086 protein,respectively. The P2086 protein of the invention may be recombinant.“rLP2086” and “rP2086” typically refer to lipidated and non-lipidatedforms of a recombinant 2086 protein, respectively. “2086” is also knownas factor H-binding protein (fHBP) due to its ability to bind to factorH.

The term “pharmaceutically acceptable carrier” as used herein isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with administration to humans or othervertebrate hosts. Typically, a pharmaceutically acceptable carrier is acarrier approved by a regulatory agency of a Federal, a stategovernment, or other regulatory agency, or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans as well as non-human mammals. The term“carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the pharmaceutical composition is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin.Water, saline solutions and aqueous dextrose and glycerol solutions canbe employed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting, bulking, emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, sustained release formulations and thelike. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. The formulationshould suit the mode of administration. The appropriate carrier will beevident to those skilled in the art and will depend in large part uponthe route of administration.

A “protective” immune response refers to the ability of an immunogeniccomposition to elicit an immune response, either humoral or cellmediated, which serves to protect the subject from an infection. Theprotection provided need not be absolute, i.e., the infection need notbe totally prevented or eradicated, if there is a statisticallysignificant improvement compared with a control population of subjects,e.g. infected animals not administered the vaccine or immunogeniccomposition. Protection may be limited to mitigating the severity orrapidity of onset of symptoms of the infection. In general, a“protective immune response” would include the induction of an increasein antibody levels specific for a particular antigen in at least 50% ofsubjects, including some level of measurable functional antibodyresponses to each antigen. In particular situations, a “protectiveimmune response” could include the induction of a two fold increase inantibody levels or a four fold increase in antibody levels specific fora particular antigen in at least 50% of subjects, including some levelof measurable functional antibody responses to each antigen. In certainembodiments, opsonising antibodies correlate with a protective immuneresponse. Thus, protective immune response may be assayed by measuringthe percent decrease in the bacterial count in a serum bactericidalactivity (SBA) assay or an opsonophagocytosis assay, for instance thosedescribed below. Such assays are also known in the art. Formeningococcal vaccines, for example, the SBA assay is an establishedsurrogate for protection. In some embodiments, there is a decrease inbacterial count of at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95%or more, as compared to the bacterial count in the absence of theimmunogenic composition.

The terms “protein”, “polypeptide” and “peptide” refer to a polymer ofamino acid residues and are not limited to a minimum length of theproduct. Thus, peptides, oligopeptides, dimers, multimers, and the like,are included within the definition. Both full-length proteins andfragments thereof are encompassed by the definition. The terms alsoinclude modifications, such as deletions, additions and substitutions(generally conservative in nature, but which may be non-conservative),to a native sequence, preferably such that the protein maintains theability to elicit an immunological response within an animal to whichthe protein is administered. Also included are post-expressionmodifications, e.g. glycosylation, acetylation, lipidation,phosphorylation and the like.

The term “recombinant” as used herein refers to any protein,polypeptide, or cell expressing a gene of interest that is produced bygenetic engineering methods. The term “recombinant” as used with respectto a protein or polypeptide, means a polypeptide produced by expressionof a recombinant polynucleotide. The proteins of the present inventionmay be isolated from a natural source or produced by genetic engineeringmethods. “Recombinant,” as used herein, further describes a nucleic acidmolecule, which, by virtue of its origin or manipulation, is notassociated with all or a portion of the polynucleotide with which it isassociated in nature. The term “recombinant” as used with respect to ahost cell means a host cell which includes a recombinant polynucleotide.

The terms “stablizer” refers to a compound that binds to an antigen andmaintains the epitopes or immunoreactivity of the antigen over a periodof time. Stabilizers are known in the art. Examples of stabilizersinclude multivalent cations, for example, calcium or aluminum.

The term “subject” refers to a mammal, bird, fish, reptile, or any otheranimal. The term “subject” also includes humans. The term “subject” alsoincludes household pets. Non-limiting examples of household petsinclude: dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters,guinea pigs, ferrets, birds, snakes, lizards, fish, turtles, and frogs.The term “subject” also includes livestock animals. Non-limitingexamples of livestock animals include: alpaca, bison, camel, cattle,deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits,reindeer, yak, chickens, geese, and turkeys.

The term “mammals” as used herein refers to any mammal, such as, forexample, humans, mice, rabbits, non-human primates. In a preferredembodiment, the mammal is a human.

The terms “vaccine” or “vaccine composition”, which are usedinterchangeably, refer to pharmaceutical compositions comprising atleast one immunogenic composition that induces an immune response in asubject.

General Description

The present invention arises out of the novel discovery that particularformulations and dosing schedules of non-lipidated variants of P2086elicit higher bactericidal antibody titers than previous formulations ofP2086, as described, for example, in Fletcher et al., Infection &Immunity. Vol. 72(4):2088-2100 (2004). Alternatively, the presentinvention arises out of the novel discovery that particular formulationsand dosing schedules of non-lipidated variants of P2086 elicit higherbactericidal antibody titers than commercially available formulations oflipidated LP2086 variants. It is noted, however, that commercialformulations of lipidated LP2086 may not be presently available. Higherresponse rates (as defined by a four fold increase or greater in SBAtiters over baseline) were observed for the vaccine containing thenon-lipidated rP2086 variant compared to the lipidated rLP2086 vaccine.The formulation of the non-lipidated P2086 variant elicited bactericidalantibodies against a broader spectrum of strains, including strains withboth similar (>92% ID) and diverse (<92% ID) LP2086 sequences.

The present invention also identifies previously unidentifieddifficulties expressing non-lipidated P2086 variants and providesmethods for overcoming these difficulties and novel compositions therefrom. While plasmid constructs encoding non-lipidated P2086 variantsprovided strong expression of the non-lipidated variants, these variantswere pyruvylated on the N-terminal Cys. Pyruvylation prevents or reducesthe likelihood of manufacturing consistency or uniformity of thepolypeptides. The inventors further found that deletion of theN-terminal Cys from the non-lipidated P2086 variant sequences avoidedpyruvylation of non-lipidated P2086 variants. Attempts to overcome thepyruvylation by deletion of the codon for the N-terminal Cys eitherabrogated expression or resulted in the expression of insolublevariants. Alternatively, removal of the N-terminal Cys from thenon-lipidated P2086 variants decreased expression in some variants.Surprisingly, however, the inventors discovered that at leastnon-pyruvylated non-lipidated A05, B01, B09, and B44 variants can beexpressed despite deletion of the N-terminal Cys residue. Generally,these polypeptides could be expressed without additional modificationsother than the Cys deletion, as compared to the corresponding wild-typenon-lipidated sequence. See, for example, Examples 2 and 4. Furthermore,the inventors discovered that the non-pyruvylated non-lipidated variantswere surprisingly immunogenic and they unexpectedly elicitedbactericidal antibodies.

Accordingly, the present invention provides two methods for overcomingor reducing the likelihood of these difficulties in expressingnon-lipidated variants. However, additional methods are contemplated bythe present invention. The first method was to vary the length of theGly/Ser stalk in the N-terminal tail, immediately downstream of theN-terminal Cys. The second method was codon optimization within theN-terminal tail. However, optimization of additional codons iscontemplated by the present invention. These methods provide enhancedexpression of soluble non-lipidated P2086 variants. For example, in oneembodiment, enhanced expression of soluble non-lipidated P2086 variantsis compared to expression of the corresponding wild-type non-lipidatedvariants.

Isolated Polypeptides

The inventors surprisingly discovered isolated non-pyruvylated,non-lipidated ORF2086 polypeptides. The inventors further discoveredthat the polypeptides are unexpectedly immunogenic and are capable ofeliciting a bactericidal immune response.

As used herein, the term “non-pyruvylated” refers to a polypeptidehaving no pyruvate content. Non-lipidated ORF2086 polypeptides having apyruvate content typically exhibited a mass shift of +70, as compared tothe corresponding wild-type polypeptide. In one embodiment, theinventive polypeptide does not exhibit a mass shift of +70 as comparedto the corresponding wild-type non-lipidated polypeptide when measuredby mass spectrometry. See, for example, Example 10.

In another embodiment, the isolated non-pyruvylated, non-lipidatedORF2086 polypeptide includes a deletion of an N-terminal cysteineresidue compared to the corresponding wild-type non-lipidated ORF2086polypeptide. The term “N-terminal cysteine” refers to a cysteine (Cys)at the N-terminal or N-terminal tail of a polypeptide. Morespecifically, the “N-terminal cysteine” as used herein refers to theN-terminal cysteine at which LP2086 lipoproteins are lipidated with atripalmitoyl lipid tail, as is known in the art. For example, whenreferring to any one of SEQ ID NOs: 12-21 as a reference sequence, theN-terminal cysteine is located at position 1.

The term “wild-type non-lipidated ORF2086 polypeptide” or “wild-typenon-lipidated 2086 polypeptide” or “wild-type non-lipidated polypeptide”as used herein refers to an ORF2086 polypeptide having an amino acidsequence that is identical to the amino acid sequence of thecorresponding mature lipidated ORF2086 polypeptide found in nature. Theonly difference between the non-lipidated and lipidated molecules isthat the wild-type non-lipidated ORF2086 polypeptide is not lipidatedwith a tripalmitoyl lipid tail at the N-terminal cysteine.

As is known in the art, the non-lipidated 2086 form is produced by aprotein lacking the original leader sequence or by a leader sequencewhich is replaced with a portion of sequence that does not specify asite for fatty acid acylation in a host cell. See, for example,WO2003/063766, which is incorporated herein by reference in itsentirety.

Examples of a non-lipidated ORF2086 include not only a wild-typenon-lipidated ORF2086 polypeptide just described but also polypeptideshaving an amino acid sequence according to any one of SEQ ID NOs: 12-21wherein the N-terminal Cys is deleted and polypeptides having an aminoacid sequence according to any one of SEQ ID NOs: 12-21 wherein theN-terminal Cys is substituted. Further examples of a non-lipidatedORF2086 polypeptide include amino acid sequences selected from SEQ IDNO: 44, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 62, andSEQ ID NO: 64.

Examples of a wild-type non-lipidated ORF2086 polypeptide includepolypeptides having an amino acid sequence according to any one of SEQID NOs: 12-21, shown in FIG. 2, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ IDNO: 60. These exemplary wild-type non-lipidated ORF2086 polypeptidesinclude an N-terminal Cys.

As used herein, for example, a “non-lipidated” B44 polypeptide includesa polypeptide having the amino acid sequence selected from SEQ ID NO:21, SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted,and SEQ ID NO: 44. A “wild-type non-lipidated” B44 polypeptide includesa polypeptide having the amino acid sequence SEQ ID NO: 21. A“non-pyruvylated non-lipidated” B44 polypeptide includes a polypeptidehaving the amino acid sequence selected from SEQ ID NO: 21 wherein theN-terminal Cys at position 1 is deleted and SEQ ID NO: 44.

As another example, as used herein, a “non-lipidated” B09 polypeptideincludes a polypeptide having the amino acid sequence selected from SEQID NO: 18, SEQ ID NO: 18 wherein the N-terminal Cys at position 1 isdeleted, SEQ ID NO: 49, and SEQ ID NO: 50. A “wild-type non-lipidated”B09 polypeptide includes a polypeptide having the amino acid sequenceSEQ ID NO: 18. A “non-pyruvylated non-lipidated” B09 includes apolypeptide having the amino acid sequence selected from SEQ ID NO: 18wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 49, andSEQ ID NO: 50.

As yet a further example, as used herein, a “non-lipidated” A05polypeptide includes a polypeptide having the amino acid sequenceselected from SEQ ID NO: 13, SEQ ID NO: 13 wherein the N-terminal Cys atposition 1 is deleted, and SEQ ID NO: 55. A “wild-type non-lipidated”A05 includes a polypeptide having the amino acid sequence SEQ ID NO: 13.A “non-pyruvylated non-lipidated” A05 includes a polypeptide having theamino acid sequence selected from SEQ ID NO: 13 wherein the N-terminalCys at position 1 is deleted and SEQ ID NO: 55.

The term “deletion” of the N-terminal Cys as used herein includes amutation that deletes the N-terminal Cys, as compared to a wild-typenon-lipidated polypeptide sequence. For example, a “deletion” of theN-terminal Cys refers to a removal of the amino acid Cys from areference sequence, e.g., from the corresponding wild-type sequence,thereby resulting in a decrease of an amino acid residue as compared tothe reference sequence.

In another embodiment, the N-terminal Cys is substituted with an aminoacid that is not a Cys residue. For example, in an exemplary embodiment,the N-terminal Cys at position 1 of SEQ ID NOs: 12-21 includes a C→Gsubstitution at position 1. See, for example, SEQ ID NO: 62 as comparedto SEQ ID NO: 19 (B22 wild-type), and SEQ ID NO: 64 as compared to SEQID NO: 15 (A22 wild-type). Exemplary amino acids to replace theN-terminal Cys include any non-Cys amino acid, preferably a polaruncharged amino acid such as, for example, glycine. In a preferredembodiment, the substitution is made with a non-conservative residue toCys.

The inventors surprisingly discovered that expressing non-lipidatedORF2086 polypeptides having a deletion of an N-terminal Cys residueresulted in no detectable pyruvylation when measured by massspectrometry, as compared to the corresponding wild-type non-lipidatedORF2086 polypeptide. Examples of non-pyruvylated non-lipidated ORF2086polypeptides include those having an amino acid sequence selected fromthe group consisting of SEQ ID NO:12 (A04), SEQ ID NO:13 (A05), SEQ IDNO:14 (A12), SEQ ID NO:15 (A22), SEQ ID NO:16 (B02) SEQ ID NO:17 (B03),SEQ ID NO:18 (B09), SEQ ID NO:19 (B22), SEQ ID NO: 20 (B24), and SEQ IDNO: 21 (B44), wherein the cysteine at position 1 is deleted. Additionalexamples of isolated non-pyruvylated, non-lipidated ORF2086 polypeptidesinclude polypeptides having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 50, and SEQID NO: 55. Preferably, the non-pyruvylated non-lipidated 2086polypeptide includes at least about 250, 255, or 260 consecutive aminoacids, and at most about 270, 269, 268, 267, 266, 265, 264, 263, 260,259, 258, 257, 256, or 255 consecutive amino acids. Any minimum valuemay be combined with any maximum value to define a range. Morepreferably, the polypeptide has at least 254 or 262 consecutive aminoacids.

In one embodiment, the isolated non-pyruvylated, non-lipidated ORF2086polypeptide is encoded by a nucleotide sequence that is operativelylinked to an expression system, wherein the expression system is capableof being expressed in a bacterial cell. In an exemplary embodiment, thenucleotide sequence is linked to a regulatory sequence that controlsexpression of the nucleotide sequence.

Suitable expression systems, regulatory sequences, and bacterial cellsare known in the art. For example, any plasmid expression vector, e.g.,PET™ (Novogen, Madison Wis.) or PMAL™ (New England Biolabs, Beverly,Mass.) can be used as long as the polypeptide is able to be expressed ina bacterial cell. Preferably, the PET™ vector is used for cloning andexpression of recombinant proteins in E. coli. In the PET™ system, thecloned gene may be expressed under the control of a phage T7 promotor.Exemplary bacterial cells include Pseudomonas fluorescens, andpreferably, E. coli.

In one aspect, the invention relates to a non-pyruvylated non-lipidatedORF2086 polypeptide obtainable by the process. The polypeptide ispreferably isolated. The invention further relates to compositions thatinclude a non-pyruvylated non-lipidated ORF2086 polypeptide obtainableby a process. The composition is preferably an immunogenic composition.The process includes expressing a nucleotide sequence encoding apolypeptide having the amino acid sequence selected from the groupconsisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16 SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO: 20,and SEQ ID NO: 21, wherein the cysteine at position 1 is deleted. Thenucleotide sequence is operatively linked to an expression system thatis capable of being expressed in a bacterial cell. In one embodiment,the process includes expressing a nucleotide sequence encoding apolypeptide having the amino acid sequence selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ IDNO: 55. In another embodiment, the nucleotide sequence is selected fromthe group consisting of SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 46, SEQID NO: 47, SEQ ID NO: 48, SEQ ID NO: 45, SEQ ID NO: 54. Preferably thebacterial cell is E. coli.

In one aspect, the invention relates to a composition that includes afirst isolated polypeptide, which includes the amino acid sequence setforth in SEQ ID NO: 49, and a second isolated polypeptide, whichincludes the amino acid sequence set forth in SEQ ID NO: 44. In apreferred embodiment, the polypeptides are immunogenic. In anotherpreferred embodiment, the composition further includes an ORF2086subfamily A polypeptide from serogroup B N. meningitidis. Preferably,the ORF2086 subfamily A polypeptide is a non-pyruvylated non-lipidatedORF2086 subfamily A polypeptide. In an exemplary embodiment, the ORF2086subfamily A polypeptide is A05, examples of which include, for example,SEQ ID NO: 13, wherein the N-terminal cysteine at position 1 is deleted,and SEQ ID NO: 55.

In another aspect, the invention relates to a method for producing anisolated polypeptide. The method includes expressing in a bacterial cella polypeptide, which includes a sequence having greater than 90%identity to SEQ ID NO:21, said sequence includes at least one domainselected from the group consisting of amino acids 13-18 of SEQ ID NO:21, amino acids 21-34 of SEQ ID NO: 21, and amino acids 70-80 of SEQ IDNO: 21, or a combination thereof, wherein the polypeptide lacks anN-terminal cysteine. The method further includes purifying thepolypeptide. The polypeptide produced therein includes a non-pyruvylatednon-lipidated ORF2086 polypeptide. Preferably, the polypeptide isimmunogenic. In a preferred embodiment, the bacterial cell is E. coli.

Examples of polypeptides that include at least one domain selected fromthe group consisting of amino acids 13-18 of SEQ ID NO: 21, amino acids21-34 of SEQ ID NO: 21, and amino acids 70-80 of SEQ ID NO: 21, or acombination thereof, include SEQ ID NO: 12 (A04), SEQ ID NO: 13 (A05),SEQ ID NO: 14 (A12), SEQ ID NO: 15 (A22), SEQ ID NO: 16 (B02), SEQ IDNO: 17 (B03), SEQ ID NO: 18 (B09), SEQ ID NO: 19 (B22), SEQ ID NO: 20(B24), and SEQ ID NO: 21 (B44). Preferably the cysteine at position 1 ofthese polypeptides is deleted. Further exemplary polypeptides includeSEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO:62, and SEQ ID NO: 64.

In one exemplary embodiment, the isolated polypeptide sequence furtherincludes at least one domain selected from the group consisting of aminoacids 96-116 of SEQ ID NO: 21, amino acids 158-170 of SEQ ID NO: 21,amino acids 172-185 of SEQ ID NO: 21, amino acids 187-199 of SEQ ID NO:21, amino acids 213-224 of SEQ ID NO: 21, amino acids 226-237 of SEQ IDNO: 21, amino acids 239-248 of SEQ ID NO: 21, or a combination thereof.Examples of polypeptides that include at least one domain selected fromthe group consisting of amino acids 13-18 of SEQ ID NO: 21, amino acids21-34 of SEQ ID NO: 21, and amino acids 70-80 of SEQ ID NO: 21, or acombination thereof, and further including at least one domain selectedfrom the group consisting of amino acids 96-116 of SEQ ID NO: 21, aminoacids 158-170 of SEQ ID NO: 21, amino acids 172-185 of SEQ ID NO: 21,amino acids 187-199 of SEQ ID NO: 21, amino acids 213-224 of SEQ ID NO:21, amino acids 226-237 of SEQ ID NO: 21, amino acids 239-248 of SEQ IDNO: 21, or a combination thereof, include SEQ ID NO: 16 (B02), SEQ IDNO: 17 (B03), SEQ ID NO: 18 (B09), SEQ ID NO: 19 (B22), SEQ ID NO: 20(B24), and SEQ ID NO: 21 (B44). Preferably the cysteine at position 1 ofthese polypeptides is deleted. Further exemplary polypeptides include apolypeptide having the amino acid sequence selected from SEQ ID NO: 44,SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 55, and SEQ ID NO: 62.

In one aspect, the invention relates to an isolated polypeptide producedby a process described herein. In one embodiment, the isolatedpolypeptide is a non-pyruvylated non-lipidated polypeptide. In anotheraspect, the invention relates to an immunogenic composition produced bya process described herein.

In one aspect, the invention relates to an isolated polypeptide thatincludes the amino acid sequence set forth in SEQ ID NO: 18 wherein theN-terminal Cys at position 1 is deleted or SEQ ID NO: 49. Exemplarynucleotide sequences that encode SEQ ID NO: 49 include sequencesselected from SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.Preferably, the nucleotide sequence is SEQ ID NO: 46. In one aspect, theinvention relates to an isolated nucleotide sequence that includes SEQID NO: 46. In one aspect, the invention relates to an isolatednucleotide sequence that includes SEQ ID NO: 47. In one aspect, theinvention relates to an isolated nucleotide sequence that includes SEQID NO: 48.

In one aspect, the invention relates to a plasmid including a nucleotidesequence selected from SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, andSEQ ID NO: 45, wherein the plasmid is capable of being expressed in abacterial cell. Suitable expression systems, regulatory sequences, andbacterial cells are known in the art, as described above. Preferably,the bacterial cell is E. coli.

In another aspect, the invention relates to an isolated polypeptide thatincludes the amino acid sequence set forth in SEQ ID NO: 50. In anexemplary embodiment, SEQ ID NO: 50 is encoded by SEQ ID NO: 45.

In yet another aspect, the invention relates to an isolated polypeptidethat includes the amino acid sequence set forth in SEQ ID NO: 21 whereinthe N-terminal Cys is deleted or SEQ ID NO: 44. Exemplary nucleotidesequences that encode SEQ ID NO: 44 include sequences selected from SEQID NO: 43 and SEQ ID NO: 51. Preferably, the nucleotide sequence is SEQID NO: 43. In one aspect, the invention relates to an isolatednucleotide sequence that includes SEQ ID NO: 43.

Immunogenic Compositions

In a preferred embodiment, the compositions described herein includingan isolated non-pyruvylated non-lipidated ORF2086 polypeptide areimmunogenic. Immunogenic compositions that include a protein encoded bya nucleotide sequence from Neisseria meningitidis ORF2086 are known inthe art. Exemplary immunogenic compositions include those described inWO2003/063766, and US patent application publication numbers US20060257413 and US 20090202593, which are incorporated herein byreference in their entirety. Such immunogenic compositions describedtherein include a protein exhibiting bactericidal activity identified asORF2086 protein, immunogenic portions thereof, and/or biologicalequivalents thereof. The ORF2086 protein refers to a protein encoded byopen reading frame 2086 of Neisseria species.

The protein may be a recombinant protein or an isolated protein fromnative Neisseria species. For example, Neisseria ORF2086 proteins may beisolated from bacterial strains, such as those of Neisseria species,including strains of Neisseria meningitidis (serogroups A, B, C, D,W-135, X, Y, Z, and 29E), Neisseria gonorrhoeae, and Neisserialactamica, as well as immunogenic portions and/or biological equivalentsof said proteins.

The ORF2086 proteins include 2086 Subfamily A proteins and Subfamily Bproteins, immunogenic portions thereof, and/or biological equivalentsthereof. 2086 subfamily A proteins and 2086 subfamily B proteins areknown in the art, see, for example Fletcher et al., 2004 cited above andMurphy et al., J Infect Dis. 2009 Aug. 1; 200(3):379-89. See alsoWO2003/063766, which discloses SEQ ID NOs: 260 to 278 therein asrepresenting amino acid sequences associated with proteins of 2086Subfamily A. In addition, disclosed in WO2003/063766 are SEQ ID NOS: 279to 299 therein as representing amino acid sequences associated withproteins of 2086 Subfamily B. WO2003/063766 is incorporated herein byreference in its entirety. The ORF2086 proteins or equivalents thereof,etc. may be lipidated or non lipidated. Preferably, the NeisseriaORF2086 protein is non lipidated. Alternatively, the immunogeniccompositions may be combinations of lipidated and non lipidated ORF2086proteins.

In (an) one embodiment, the immunogenic composition includes an isolatedprotein having at least 95% amino acid sequence identity to a proteinencoded by a nucleotide sequence from Neisseria ORF2086.

In one embodiment, the immunogenic composition includes an isolatedprotein having at least 95% amino acid sequence identity to a SubfamilyA protein encoded by a nucleotide sequence from Neisseria ORF2086.Preferably, the immunogenic composition includes an isolated Subfamily Aprotein encoded by a nucleotide sequence from Neisseria ORF2086. In someembodiments, the ORF2086 Subfamily A polypeptide is an A05, an A04, anA12, or an A22 variant. In some embodiments, the ORF2086 Subfamily Apolypeptide is an A05, an A12, or an A22 variant.

In another embodiment, the immunogenic composition includes an isolatedprotein having at least 95% amino acid sequence identity to a SubfamilyB protein encoded by a nucleotide sequence from Neisseria ORF2086.Preferably, the immunogenic composition includes an isolated Subfamily Bprotein encoded by a nucleotide sequence from Neisseria ORF2086. In someembodiments, the ORF2086 Subfamily B protein is a B44, a B02, a B03, aB22, a B24 or a B09 variant. In some embodiments, the ORF2086 SubfamilyB protein is a B44, a B22, or a B09 variant.

In a preferred embodiment, the immunogenic composition includes anisolated non-pyruvylated non-lipidated polypeptide having at least 95%amino acid sequence identity to a Subfamily B protein encoded by anucleotide sequence from Neisseria ORF2086. For example, in someembodiments, the ORF2086 Subfamily B protein is sequences selected froma B44 having an amino acid sequence as shown in SEQ ID NO: 21; a B02having an amino acid sequence as shown in SEQ ID NO: 16; a B03 having anamino acid sequence as shown in SEQ ID NO: 17; a B22 having an aminoacid sequence as shown in SEQ ID NO:19; a B24 having an amino acidsequence as shown in SEQ ID NO: 20; or a B09 variant having an aminoacid sequence as shown in SEQ ID NO:18, wherein the N-terminal Cys isdeleted, ora combination thereof.

More preferably, the immunogenic composition includes a non-pyruvylatednon-lipidated B09 polypeptide, a non-pyruvylated non-lipidated B44polypeptide, or combinations thereof. In one embodiment, the compositionincludes a non-pyruvylated non-lipidated B09 variant having the aminoacid sequence as shown in SEQ ID NO:18, wherein the N-terminal Cys isdeleted, a non-pyruvylated non-lipidated B44 having the amino acidsequence as shown in SEQ ID NO: 21, wherein the N-terminal Cys isdeleted, or a combination thereof. In another embodiment, theimmunogenic composition includes a non-pyruvylated non-lipidated B09having SEQ ID NO: 49, a non-pyruvylated non-lipidated B44 having SEQ IDNO: 44, or a combination thereof.

In one aspect, the invention relates to an immunogenic composition thatincludes an ORF2086 subfamily B polypeptide from serogroup B N.meningitidis, wherein the polypeptide is a non-pyruvylated non-lipidatedB44. The B44 may include the amino acid sequence as shown in SEQ ID NO:21, wherein the N-terminal Cys is deleted or SEQ ID NO: 44. In oneembodiment, the composition further includes a second ORF2086 subfamilyB polypeptide from serogroup B N. meningitidis, wherein the secondpolypeptide is a non-pyruvylated non-lipidated B09. The B09 may includethe amino acid sequence as shown in SEQ ID NO: 18, wherein theN-terminal Cys is deleted, or SEQ ID NO: 49. In one embodiment, theimmunogenic composition is a vaccine.

In another embodiment, the composition includes no more than 3 ORF2086subfamily B polypeptides. In a further embodiment, the compositionincludes no more than 2 ORF2086 subfamily B polypeptides.

In one embodiment, the composition further includes one or more ORF2086subfamily A polypeptides. In a preferred embodiment, the compositionincludes an A05 subfamily A polypeptide.

In yet another embodiment, the immunogenic composition includes anisolated protein having at least 95% amino acid sequence identity to aSubfamily A protein encoded by a nucleotide sequence from NeisseriaORF2086, and an isolated protein having at least 95% amino acid sequenceidentity to a Subfamily B protein encoded by a nucleotide sequence fromNeisseria ORF2086.

Preferably, the immunogenic composition includes an isolated Subfamily Aprotein encoded by a nucleotide sequence from Neisseria ORF2086 and anisolated Subfamily B protein encoded by a nucleotide sequence fromNeisseria ORF2086. More preferably, the immunogenic composition includesan isolated non-pyruvylated non-lipidated Subfamily A ORF2086polypeptide and an isolated non-pyruvylated non-lipidated Subfamily BORF2086 polypeptide. In some embodiments, the ORF2086 Subfamily Apolypeptide is an A05, an A04, an A12, or an A22 variant. In a preferredembodiment, the ORF2086 Subfamily A polypeptide is an A05 having anamino acid sequence as shown in SEQ ID NO: 13; an A04 having an aminoacid sequence as shown in SEQ ID NO: 12; an A12 having an amino acidsequence as shown in SEQ ID NO: 14; or an A22 variant having an aminoacid sequence as shown in SEQ ID NO: 15, wherein the N-terminal Cys isdeleted, or any combination thereof. In some embodiments, the ORF2086Subfamily B protein is a B44, a B02, a B03, a B22, a B24 or a B09variant. In a preferred embodiment, the ORF2086 Subfamily B protein is aB44 having the amino acid sequence as shown in SEQ ID NO: 21; a B02having an amino acid sequence as shown in SEQ ID NO: 16; a B03 having anamino acid sequence as shown in SEQ ID NO: 17; a B22 having an aminoacid sequence as shown in SEQ ID NO:19; a B24 having an amino acidsequence as shown in SEQ ID NO: 20; ora B09 variant having an amino acidsequence as shown in SEQ ID NO:18, wherein the N-terminal Cys isdeleted, or a combination thereof.

In one embodiment, the immunogenic composition includes a 1:1 ratio of aSubfamily A protein to a Subfamily B protein.

In another aspect, the isolated polypeptides and compositions describedherein elicit a bactericidal immune response in a mammal against anORF2086 polypeptide from serogroup B N. meningitidis. The compositionshave the ability to induce bactericidal anti-meningococcal antibodiesafter administration to a mammal, and in preferred embodiments caninduce antibodies that are bactericidal against strains with therespective subfamilies. Further information on bactericidal responses isgiven below. See, for example, Examples 6, 11, 12, and 13. Bactericidalantibodies are an indicator of protection in humans and preclinicalstudies serve as a surrogate, and any new immunogenic compositioncandidate should elicit these functional antibodies.

In an exemplary embodiment, the isolated non-pyruvylated non-lipidatedB09 polypeptide having SEQ ID NO: 18 wherein the N-terminal Cys atposition 1 is deleted or SEQ ID NO: 49, and immunogenic compositionsthereof, elicits bactericidal antibodies against (e.g., that can bindto) an ORF2086 polypeptide from serogroup B N. meningitidis, subfamily Aor preferably subfamily B. Preferably, the non-pyruvylated non-lipidatedB09 polypeptide and immunogenic compositions thereof, elicitsbactericidal antibodies against the A05 variant (SEQ ID NO: 13); B44variant (SEQ ID NO: 21); B16 variant (SEQ ID NO: 60); B24 variant (SEQID NO: 20); B09 variant (SEQ ID NO: 18), ora combination thereof. In anexemplary embodiment, the non-pyruvylated non-lipidated B09 polypeptideand immunogenic compositions thereof, elicits bactericidal antibodiesagainst B44 variant (SEQ ID NO: 21); B16 variant (SEQ ID NO: 60); B24variant (SEQ ID NO: 20); B09 variant (SEQ ID NO: 18), or a combinationthereof. See, for example, Example 11, Example 12, and Example 13.

In another exemplary embodiment, the isolated non-pyruvulatednon-lipidated B44 polypeptide having SEQ ID NO: 21 wherein theN-terminal Cys at position 1 is deleted or SEQ ID NO: 44, andimmunogenic compositions thereof, elicits bactericidal antibodiesagainst (e.g., that can bind to) an ORF2086 polypeptide from serogroup BN. meningitidis, subfamily B. Preferably, the non-pyruvylatednon-lipidated B44 polypeptide and immunogenic compositions thereof,elicits bactericidal antibodies against the B44 variant (SEQ ID NO: 21);B16 variant (SEQ ID NO: 60); B24 variant (SEQ ID NO: 20); B09 variant(SEQ ID NO: 18), or a combination thereof. See, for example, Example 11.Additionally, the non-pyruvylated non-lipidated B44 polypeptide andimmunogenic compositions thereof may also elicit bactericidal antibodiesthat bind to the B02 variant (SEQ ID NO: 16). See, for example, Example12 and Example 13. Moreover, the non-pyruvylated non-lipidated B44polypeptide and immunogenic compositions thereof may also elicitbactericidal antibodies that bind to B03 variant (SEQ ID NO: 17) and B15variant (SEQ ID NO: 59). See, for example, Example 6.

In a further exemplary embodiment, the isolated non-pyruvulatednon-lipidated B22 polypeptide having SEQ ID NO: 19 wherein theN-terminal Cys at position 1 is deleted, and immunogenic compositionsthereof, elicits bactericidal antibodies against (e.g., that can bindto) an ORF2086 polypeptide from serogroup B N. meningitidis, subfamilyB. Preferably, the non-pyruvylated non-lipidated B22 polypeptide elicitsbactericidal antibodies against the B44 variant (SEQ ID NO: 21); B16variant (SEQ ID NO: 60); B24 variant (SEQ ID NO: 20); B09 variant (SEQID NO: 18), or a combination thereof. See, for example, Example 13.

In one embodiment, the isolated non-pyruvylated non-lipidated A05polypeptide having SEQ ID NO: 13 wherein the N-terminal Cys is deletedor SEQ ID NO: 55, and immunogenic compositions thereof, elicitsbacteridial antibodies against (e.g., that can bind to) an ORF2086polypeptide from serogroup B N. meningitidis, subfamily A. Preferably,the non-pyruvylated non-lipidated A05 and immunogenic compositionsthereof, elicits bactericidal antibodies against the A05 variant (SEQ IDNO: 13), A22 variant (SEQ ID NO: 15), A12 variant (SEQ ID NO: 14), oracombination thereof. See, for example, Example 6 and 13.

In one aspect, the invention relates to a method of elicitingbactericidal antibodies specific to serogroup B N. meningitidis in amammal. In an exemplary embodiment, the method includes elicitingbactericidal antibodies specific to an ORF2086 subfamily B serogroup BN. meningitidis, an ORF2086 subfamily A serogroup B N. meningitidis, ora combination thereof. The method includes administering to the mammalan effective amount of an isolated non-pyruvylated non-lipidated 2086polypeptide or immunogenic composition thereof, as described above.

In a preferred embodiment, the method includes eliciting bactericidalantibodies specific to an ORF2086 subfamily B serogroup B N.meningitidis. The isolated polypeptide or immunogenic compositionincludes a non-pyruvylated non-lipidated B44 polypeptide. In anotherpreferred embodiment, the composition further includes a non-pyruvylatednon-lipidated B09 polypeptide. In an exemplary embodiment, the isolatedpolypeptide or immunogenic composition includes SEQ ID NO: 49, SEQ IDNO: 44, or a combination thereof. In another exemplary embodiment, theisolated polypeptide or immunogenic composition includes SEQ ID NO: 18,wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 21,wherein the N-terminal Cys at position 1 is deleted, or a combinationthereof, In yet another exemplary embodiment, the isolated polypeptideor immunogenic composition includes SEQ ID NO: 19, wherein theN-terminal Cys at position 1 is deleted,

In a preferred embodiment, the method includes eliciting bactericidalantibodies specific to an ORF2086 subfamily A serogroup B N.meningitidis. The isolated polypeptide or immunogenic compositionincludes a non-pyruvylated non-lipidated A05 polypeptide. In a preferredembodiment, the isolated polypeptide or immunogenic composition includesSEQ ID NO: 13, wherein the N-terminal Cys at position 1 is deleted, Inanother preferred embodiment, the composition further includes anon-pyruvylated non-lipidated B44 polypeptide. See, for example, Example6 and 13. In an exemplary embodiment, the isolated polypeptide orimmunogenic composition includes SEQ ID NO: 55, SEQ ID NO: 44, or acombination thereof. In a preferred embodiment, the isolated polypeptideor immunogenic composition includes SEQ ID NO: 13, wherein theN-terminal Cys at position 1 is deleted, SEQ ID NO: 21, wherein theN-terminal Cys at position 1 is deleted, or a combination thereof.

The immunogenic composition may include a protein encoded by anucleotide sequence from Neisseria ORF2086, polynucleotides, orequivalents thereof as the sole active immunogen in the immunogeniccomposition. Alternatively, the immunogenic composition may furtherinclude active immunogens, including other Neisseria sp. immunogenicpolypeptides, or immunologically-active proteins of one or more othermicrobial pathogens (e.g. virus, prion, bacterium, or fungus, withoutlimitation) or capsular polysaccharide. The compositions may compriseone or more desired proteins, fragments or pharmaceutical compounds asdesired for a chosen indication.

Any multi-antigen or multi-valent immunogenic composition iscontemplated by the present invention. For example, the immunogeniccomposition may include combinations of two or more ORF2086 proteins, acombination of ORF2086 protein with one or more Por A proteins, acombination of ORF2086 protein with meningococcus serogroup A, C, Y andW135 polysaccharides and/or polysaccharide conjugates, a combination ofORF2086 protein with meningococcus and pneumococcus combinations, or acombination of any of the foregoing in a form suitable for a desiredadministration, e.g., for mucosal delivery. Persons of skill in the artwould be readily able to formulate such multi-antigen or multi-valentimmunologic compositions.

The present invention also contemplates multi-immunization regimenswherein any composition useful against a pathogen may be combinedtherein or therewith the compositions of the present invention. Forexample, without limitation, a patient may be administered theimmunogenic composition of the present invention and anotherimmununological composition for immunizing against human papillomavirusvirus (HPV), such as the HPV vaccine GARDASIL®, as part of amulti-immunization regimen. Persons of skill in the art would be readilyable to select immunogenic compositions for use in conjunction with theimmunogenic compositions of the present invention for the purposes ofdeveloping and implementing multi-immunization regimens.

The ORF2086 polypeptides, fragments and equivalents can be used as partof a conjugate immunogenic composition; wherein one or more proteins orpolypeptides are conjugated to a carrier in order to generate acomposition that has immunogenic properties against several serotypes,or serotypes of N. meningitidis, specifically meningococcus serogroupsspecifically serogroup B, and/or against several diseases.Alternatively, one of the ORF2086 polypeptides can be used as a carrierprotein for other immunogenic polypeptides. Formulation of suchimmunogenic compositions is well known to persons skilled in this field.

Immunogenic compositions of the invention preferably include apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers and/or diluents include any and all conventionalsolvents, dispersion media, fillers, solid carriers, aqueous solutions,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. Suitable pharmaceutically acceptablecarriers include, for example, one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof.

Pharmaceutically acceptable carriers may further include minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody. The preparation and use of pharmaceutically acceptablecarriers is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the immunogenic compositions of the present invention iscontemplated.

Immunogenic compositions can be administered parenterally, e.g., byinjection, either subcutaneously or intramuscularly, as well as orallyor intranasally. Methods for intramuscular immunization are described byWolff et al. Biotechniques; 11(4):474-85, (1991). and by Sedegah et al.PNAS Vol. 91, pp. 9866-9870, (1994). Other modes of administrationemploy oral formulations, pulmonary formulations, suppositories, andtransdermal applications, for example, without limitation. Oralformulations, for example, include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,and the like, without limitation. Preferably, the immunogeniccomposition is administered intramuscularly.

The immunogenic compositions of the present invention can furthercomprise one or more additional “immunomodulators”, which are agentsthat perturb or alter the immune system, such that either up-regulationor down-regulation of humoral and/or cell-mediated immunity is observed.In one particular embodiment, up-regulation of the humoral and/orcell-mediated arms of the immune system is preferred. Examples ofcertain immunomodulators include, for example, an adjuvant or cytokine,or ISCOMATRIX (CSL Limited, Parkville, Australia), described in U.S.Pat. No. 5,254,339 among others.

Non-limiting examples of adjuvants that can be used in the vaccine ofthe present invention include the RIBI adjuvant system (Ribi Inc.,Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel,oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund'scomplete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.),QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron,Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponinfraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant.Non-limiting examples of oil-in-water emulsions useful in the vaccine ofthe invention include modified SEAM62 and SEAM 1/2 formulations.Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene(Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v)polysorbate ® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. ModifiedSEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1%(v/v) SPAN® 85 detergent, 0.7% (v/v) polysorbate 80 detergent, 2.5%(v/v) ethanol, 100 μg/ml Quil A, and 50 μg/ml cholesterol.

Other “immunomodulators” that can be included in the vaccine include,e.g., one or more interleukins, interferons, or other known cytokines orchemokines. In one embodiment, the adjuvant may be a cyclodextrinderivative or a polyanionic polymer, such as those described in U.S.Pat. Nos. 6,165,995 and 6,610,310, respectively. It is to be understoodthat the immunomodulator and/or adjuvant to be used will depend on thesubject to which the vaccine or immunogenic composition will beadministered, the route of injection and the number of injections to begiven.

In some embodiments, the adjuvant is saponin. In some embodiments, thesaponin concentration is between 1 μg/ml and 250 μg/ml, between 5 μg/mland 150 μg/ml, or between 10 μg/ml and 100 μg/ml. In some embodiments,the saponin concentration is about 1 μg/ml, about 5 μg/ml, about 10μg/ml, about 20 μg/ml, about 30 μg/ml, about 40 μg/ml, about 50 μg/ml,about 60 μg/ml, about 70 μg/ml, about 80 μg/ml, about 90 μg/ml, about100 μg/ml, about 110 μg/ml, about 120 μg/ml, about 130 μg/ml, about 140μg/ml, about 150 μg/ml, about 160 μg/ml, about 170 μg/ml, about 180μg/ml, about 190 μg/ml, about 200 μg/ml, about 210 μg/ml, about 220μg/ml, about 230 μg/ml, about 240 μg/ml, or about 250 μg/ml.

In certain preferred embodiments, the proteins of this invention areused in an immunogenic composition for oral administration whichincludes a mucosal adjuvant and used for the treatment or prevention ofN. meningitidis infection in a human host. The mucosal adjuvant can be acholera toxin; however, preferably, mucosal adjuvants other than choleratoxin which may be used in accordance with the present invention includenon-toxic derivatives of a cholera holotoxin, wherein the A subunit ismutagenized, chemically modified cholera toxin, or related proteinsproduced by modification of the cholera toxin amino acid sequence. For aspecific cholera toxin which may be particularly useful in preparingimmunogenic compositions of this invention, see the mutant choleraholotoxin E29H, as disclosed in Published International Application WO00/18434, which is hereby incorporated herein by reference in itsentirety. These may be added to, or conjugated with, the polypeptides ofthis invention. The same techniques can be applied to other moleculeswith mucosal adjuvant or delivery properties such as Escherichia coliheat labile toxin (LT).

Other compounds with mucosal adjuvant or delivery activity may be usedsuch as bile; polycations such as DEAE-dextran and polyornithine;detergents such as sodium dodecyl benzene sulphate; lipid-conjugatedmaterials; antibiotics such as streptomycin; vitamin A; and othercompounds that alter the structural or functional integrity of mucosalsurfaces. Other mucosally active compounds include derivatives ofmicrobial structures such as MDP; acridine and cimetidine. STIMULON™QS-21, MPL, and IL-12, as described above, may also be used.

The immunogenic compositions of this invention may be delivered in theform of ISCOMS (immune stimulating complexes), ISCOMS containing CTB,liposomes or encapsulated in compounds such as acrylates orpoly(DL-lactide-co-glycoside) to form microspheres of a size suited toadsorption. The proteins of this invention may also be incorporated intooily emulsions.

An amount (i.e., dose) of immunogenic composition that is administeredto the patient can be determined in accordance with standard techniquesknown to those of ordinary skill in the art, taking into considerationsuch factors as the particular antigen, the adjuvant (if present), theage, sex, weight, species, condition of the particular patient, and theroute of administration.

For example, a dosage for an adolescent human patient may include atleast 0.1μg, 1 μg, 10 μg, or 50 μg of a Neisseria ORF2086 protein, andat most 80 μg, 100 μg, 150 μg, or 200 μg of a Neisseria ORF2086 protein.Any minimum value and any maximum value may be combined to define asuitable range.

Adjuvants

Immunogenic compositions as described herein also comprise, in certainembodiments, one or more adjuvants. An adjuvant is a substance thatenhances the immune response when administered together with animmunogen or antigen. A number of cytokines or lymphokines have beenshown to have immune modulating activity, and thus are useful asadjuvants, including, but not limited to, the interleukins 1-α, 1-β, 2,4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15,16, 17 and 18 (and its mutant forms); the interferons-α, β and γ;granulocyte-macrophage colony stimulating factor (GM-CSF) (see, e.g.,U.S. Pat. No. 5,078,996 and ATCC Accession Number 39900); macrophagecolony stimulating factor (M-CSF); granulocyte colony stimulating factor(G-CSF); and the tumor necrosis factors α and β.

Still other adjuvants that are useful with the immunogenic compositionsdescribed herein include chemokines, including without limitation,MCP-1, MIP-1α, MIP-1β, and RANTES; adhesion molecules, such as aselectin, e.g., L-selectin, P-selectin and E-selectin; mucin-likemolecules, e.g., CD34, GlyCAM-1 and MadCAM-1, a member of the integrinfamily such as LFA-1, VLA-1, Mac-1 and p150.95, a member of theimmunoglobulin superfamily such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2and ICAM-3, CD2 and LFA-3; co-stimulatory molecules such as B7-1,B7-2,CD40 and CD40L, growth factors including vascular growth factor,nerve growth factor, fibroblast growth factor, epidermal growth factor,PDGF, BL-1, and vascular endothelial growth factor; receptor moleculesincluding Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3,AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, and DR6; andCaspase (ICE).

Other exemplary adjuvants include, but are not limited to aluminumhydroxide; aluminum phosphate; STIMULON™ QS-21 (AquilaBiopharmaceuticals, Inc., Framingham, Mass.); MPL™ (3-O-deacylatedmonophosphoryl lipid A; Corixa, Hamilton, Mont.), 529 (an amino alkylglucosamine phosphate compound, Corixa, Hamilton, Mont.), IL-12(Genetics Institute, Cambridge, Mass.); GM-CSF (Immunex Corp., Seattle,Wash.); N-acetyl-muramyl-L-theronyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy-ethylamine) (CGP 19835A, referred to as MTP-PE); and cholera toxin. In certainpreferred embodiments, the adjuvant is QS-21.

Additional exemplary adjuvants include non-toxic derivatives of choleratoxin, including its A subunit, and/or conjugates or geneticallyengineered fusions of the N. meningitidis polypeptide with cholera toxinor its B subunit (“CTB”), procholeragenoid, fungal polysaccharides,including schizophyllan, muramyl dipeptide, muramyl dipeptide (“MDP”)derivatives, phorbol esters, the heat labile toxin of E. coli, blockpolymers or saponins.

Aluminum phosphate has been used as the adjuvant in a phase 1 clinicaltrial to a concentration 0.125 mg/dose, much lower than the limit of0.85 mg/dose specified by the US Code of Federal Regulations[610.15(a)]. Aluminum-containing adjuvants are widely used in humans topotentiate the immune response of antigens when administeredintramuscularly or subcutaneously. In some embodiments, theconcentration of aluminum in the immunogenic composition is between0.125 μg/ml and 0.5 μg/ml, between 0.20 μg/ml and 0.40 μg/ml, or between0.20 μg/ml and 0.30 μg/ml. In some embodiments, the concentration ofaluminum in the immunogenic composition is about 0.125 μg/ml, about 0.15μg/ml, about 0.175 μg/ml, about 0.20 μg/ml, about 0.225 μg/ml, about0.25 μg/ml, about 0.275 μg/ml, about 0.30 μg/ml, about 0.325 μg/ml,about 0.35 μg/ml, about 0.375 μg/ml, about 0.40 μg/ml, about 0.425μg/ml, about 0.45 μg/ml, about 0.475 μg/ml, or about 0.50 μg/ml.

In a preferred embodiment, the concentration of aluminum in theimmunogenic composition is between 0.125 mg/ml and 0.5 mg/ml, between0.20 mg/ml and 0.40 mg/ml, or between 0.20 mg/ml and 0.30 mg/ml. In someembodiments, the concentration of aluminum in the immunogeniccomposition is about 0.125 mg/ml, about 0.15 mg/ml, about 0.175 mg/ml,about 0.20 mg/ml, about 0.225 mg/ml, about 0.25 mg/ml, about 0.275mg/ml, about 0.30 mg/ml, about 0.325 mg/ml, about 0.35 mg/ml, about0.375 mg/ml, about 0.40 mg/ml, about 0.425 mg/ml, about 0.45 mg/ml,about 0.475 mg/ml, or about 0.50 mg/ml.

Suitable adjuvants used to enhance an immune response further include,without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa,Hamilton, Mont.), which is described in U.S. Pat. No. 4,912,094. Alsosuitable for use as adjuvants are synthetic lipid A analogs oraminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa (Hamilton, Mont.), andwhich are described in U.S. Pat. No. 6,113,918. One such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetrade-canoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529). This 529 adjuvantis formulated as an aqueous form (AF) or as a stable emulsion (SE).

Still other adjuvants include muramyl peptides, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryl-oxy)-ethylamine(MTP-PE); oil-in-water emulsions, such as MF59 (U.S. Pat. No. 6,299,884)(containing 5% Squalene, 0.5% polysorbate 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.)), and SAF (containing 10%Squalene, 0.4% polysorbate 80, 5% pluronic-blocked polymer L121, andthr-MDP, either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion); incomplete Freund's adjuvant(IFA); aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate; Amphigen; Avridine; L121/squalene;D-lactide-polylactide/glycoside; pluronic polyols; killed Bordetella;saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, Mass.),described in U.S. Pat. No. 5,057,540, ISCOMATRIX (CSL Limited,Parkville, Australia), described in U.S. Pat. No. 5,254,339, andimmunostimulating complexes (ISCOMATRIX), Mycobacterium tuberculosis;bacterial lipopolysaccharides, synthetic polynucleotides such asoligonucleotides containing a CpG motif (e.g., U.S. Pat. No. 6,207,646);IC-31 (Intercell AG, Vienna, Austria), described in European Patent Nos.1,296,713 and 1,326,634; a pertussis toxin (PT) or mutant thereof, acholera toxin or mutant thereof (e.g., U.S. Pat. Nos. 7,285,281,7,332,174, 7,361,355 and 7,384,640); or an E. coli heat-labile toxin(LT) or mutant thereof, particularly LT-K63, LT-R72 (e.g., U.S. Pat.Nos. 6,149,919, 7,115,730 and 7,291,588).

Methods of Producing Non-Lipidated P2086 Antigens

In one aspect, the invention relates to a method of producing anon-pyruvylated non-lipidated ORF2086 polypeptide. The method includesexpressing a nucleotide sequence encoding a ORF2086 polypeptide whereinthe N-terminal cysteine is deleted as compared to the correspondingwild-type sequence, and wherein the nucleotide sequence is operativelylinked to an expression system that is capable of being expressed in abacterial cell. Exemplary polypeptides produced by the method includeany polypeptide described herein. For example, preferably, thepolypeptide has the amino acid sequence set forth in SEQ ID NO: 12; SEQID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17;SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21, wherein thecysteine at position 1 is deleted, as compared to the correspondingwild-type sequence. Additional exemplary polypeptides include apolypeptide having the amino acid sequences sequences selected from SEQID NO: 44, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 57,SEQ ID NO: 62, and SEQ ID NO: 64. The method further includes purifyingthe polypeptide.

In some embodiments, the invention provides a method for producingsoluble non-lipidated P2086 antigens comprising the steps of cloning theORF2086 variant nucleic acid sequence into an E. coli expression vectorwithout a lipidation control sequence, transforming E. coli bacteriawith the ORF2086 expression vector, inducing expression and isolatingthe expressed P2086 protein. In some embodiments, expression is inducedwith IPTG.

In some embodiments, the codon for the N-terminal Cys of the ORF2086variant is deleted. Examples of such codons include TGC. In someembodiments, the codon for the N-terminal Cys of the ORF2086 variant ismutated by point mutagenesis to generate an Ala, Gly, or Val codon. Insome embodiments, Ser and Gly codons are added to the N-terminal tail ofthe ORF2086 variant to extend the Gly/Ser stalk immediately downstreamof the N-terminal Cys. In some embodiments, the total number of Gly andSer residues within the Gly/Ser stalk is at least 7, 8, 9, 10, 11, or12. In some embodiments, the codon for the N-terminal Cys is deleted. Insome embodiments, the N-terminal 7, 8, 9, 10, 11, or 12 residues areeither Gly or Ser.

In some embodiments, the codons of the N-terminal tail of thenon-lipidated ORF2086 variant are optimized by point mutagenesis. Insome embodiments, the N-terminal tail of the non-lipidated ORF2086variant is optimized to match the N-terminal tail of the B09 variant. Insome embodiments, the codons of the N-terminal tail of the ORF2086variant are optimized by point mutagenesis such that the codon encodingthe fifth amino acid of the ORF2086 variant is 100% identical tonucleotides 13-15 of SEQ ID NO: 8 and the codon encoding the thirteenthamino acid of the ORF2086 variant is 100% identical to nucleotides 37-39of SEQ ID NO: 8. In some embodiments, the N-terminal tail of thenon-lipidated ORF2086 variant is optimized such that the 5′ 45 nucleicacids are 100% identical to nucleic acids 1-45 of SEQ ID NO: 8. In someembodiments, the N-terminal tail of the non-lipidated ORF2086 variant isoptimized such that the 5′ 42 nucleic acids are 100% identical tonucleic acids 4-45 of SEQ ID NO: 8. In some embodiments, the N-terminaltail of the non-lipidated ORF2086 variant is optimized such that the 5′39 nucleic acids are 100% identical to nucleic acids 4-42 of SEQ ID NO:8. In some embodiments, the N-terminal tail of the non-lipidated P2086variant comprises at least one amino acid substitution compared to aminoacids 1-15 of SEQ ID NO: 18. In some embodiments, the N-terminal tail ofthe non-lipidated P2086 variant comprises two amino acid substitutionscompared to amino acids 1-15 of SEQ ID NO: 18. In some embodiments, theN-terminal tail of the non-lipidated P2086 variant comprises at leastone amino acid substitution compared to amino acids 2-15 of SEQ ID NO:18. In some embodiments, the N-terminal tail of the non-lipidated P2086variant comprises two amino acid substitutions compared to amino acids2-15 of SEQ ID NO: 18. In some embodiments, the amino acid substitutionsare conservative amino acid substitutions.

In some embodiments, the codons of the non-lipidated variant have beenoptimized for increased expression. Codon optimization is known in theart. See, e.g., Sastalla et al, Applied and Environmental Microbiology,vol. 75(7): 2099-2110 (2009) and Coleman et al, Science, vol. 320: 1784(2008). In some embodiments, codon optimization includes matching thecodon utilization of an amino acid sequence with the codon frequency ofthe host organism chosen while including and/or excluding specific DNAsequences. In some embodiments, codon optimization further includesminimizing the corresponding secondary mRNA structure to reducetranslational impediments. In some embodiments, the N-terminal tail hasbeen codon optimized to comprise any one of SEQ ID NO: 28, 30, 32, and34. In some embodiments, the Gly/Ser stalk has been codon optimized tocomprise any one of SEQ ID NO: 28, 30, 32, and 34.

In order that this invention may be better understood, the followingexamples are set forth. The examples are for the purpose of illustrationonly and are not to be construed as limiting the scope of the invention.

Immunogenic Composition Formulations

In certain embodiments, the immunogenic compositions of the inventionfurther comprise at least one of an adjuvant, a buffer, acryoprotectant, a salt, a divalent cation, a non-ionic detergent, aninhibitor of free radical oxidation, a diluent or a carrier.

The immunogenic compositions of the invention may further comprise oneor more preservatives in addition to a plurality of meningococcalprotein antigens and capsular polysaccharide-protein conjugates. The FDArequires that biological products in multiple-dose (multi-dose) vialscontain a preservative, with only a few exceptions. Vaccine productscontaining preservatives include vaccines containing benzethoniumchloride (anthrax), 2-phenoxyethanol (DTaP, HepA, Lyme, Polio(parenteral)), phenol (Pneumo, Typhoid (parenteral), Vaccinia) andthimerosal (DTaP, DT, Td, HepB, Hib, Influenza, JE, Mening, Pneumo,Rabies). Preservatives approved for use in injectable drugs include,e.g., chlorobutanol, m-cresol, methylparaben, propylparaben,2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoicacid, benzyl alcohol, phenol, thimerosal and phenylmercuric nitrate.

Formulations of the invention may further comprise one or more of abuffer, a salt, a divalent cation, a non-ionic detergent, acryoprotectant such as a sugar, and an anti-oxidant such as a freeradical scavenger or chelating agent, or any multiple combinationthereof. The choice of any one component, e.g., a chelator, maydetermine whether or not another component (e.g., a scavenger) isdesirable. The final composition formulated for administration should besterile and/or pyrogen free. The skilled artisan may empiricallydetermine which combinations of these and other components will beoptimal for inclusion in the preservative containing immunogeniccompositions of the invention depending on a variety of factors such asthe particular storage and administration conditions required.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or morephysiologically acceptable buffers selected from, but not limited to,Tris (trimethamine), phosphate, acetate, borate, citrate, glycine,histidine and succinate. In certain embodiments, the formulation isbuffered to within a pH range of about 6.0 to about 9.0, preferably fromabout 6.4 to about 7.4.

In certain embodiments, it may be desirable to adjust the pH of theimmunogenic composition or formulation of the invention. The pH of aformulation of the invention may be adjusted using standard techniquesin the art. The pH of the formulation may be adjusted to be between 3.0and 8.0. In certain embodiments, the pH of the formulation may be, ormay adjusted to be, between 3.0 and 6.0, 4.0 and 6.0, or 5.0 and 8.0. Inother embodiments, the pH of the formulation may be, or may adjusted tobe, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5,about 5.8, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. Incertain embodiments, the pH may be, or may adjusted to be, in a rangefrom 4.5 to 7.5, or from 4.5 to 6.5, from 5.0 to 5.4, from 5.4 to 5.5,from 5.5 to 5.6, from 5.6 to 5.7, from 5.7 to 5.8, from 5.8 to 5.9, from5.9 to 6.0, from 6.0 to 6.1, from 6.1 to 6.2, from 6.2 to 6.3, from 6.3to 6.5, from 6.5 to 7.0, from 7.0 to 7.5 or from 7.5 to 8.0. In aspecific embodiment, the pH of the formulation is about 5.8.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or more divalentcations, including but not limited to MgCl₂, CaCl₂ and MnCl₂, at aconcentration ranging from about 0.1 mM to about 10 mM, with up to about5 mM being preferred.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or more salts,including but not limited to sodium chloride, potassium chloride, sodiumsulfate, and potassium sulfate, present at an ionic strength which isphysiologically acceptable to the subject upon parenteral administrationand included at a final concentration to produce a selected ionicstrength or osmolarity in the final formulation. The final ionicstrength or osmolality of the formulation will be determined by multiplecomponents (e.g., ions from buffering compound(s) and othernon-buffering salts. A preferred salt, NaCl, is present from a range ofup to about 250 mM, with salt concentrations being selected tocomplement other components (e.g., sugars) so that the final totalosmolarity of the formulation is compatible with parenteraladministration (e.g., intramuscular or subcutaneous injection) and willpromote long term stability of the immunogenic components of theimmunogenic composition formulation over various temperature ranges.Salt-free formulations will tolerate increased ranges of the one or moreselected cryoprotectants to maintain desired final osmolarity levels.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or morecryoprotectants selected from but not limited to disaccharides (e.g.,lactose, maltose, sucrose or trehalose) and polyhydroxy hydrocarbons(e.g., dulcitol, glycerol, mannitol and sorbitol).

In certain embodiments, the osmolarity of the formulation is in a rangeof from about 200 mOs/L to about 800 mOs/L, with a preferred range offrom about 250 mOs/L to about 500 mOs/L, or about 300 mOs/L-about 400mOs/L. A salt-free formulation may contain, for example, from about 5%to about 25% sucrose, and preferably from about 7% to about 15%, orabout 10% to about 12% sucrose. Alternatively, a salt-free formulationmay contain, for example, from about 3% to about 12% sorbitol, andpreferably from about 4% to 7%, or about 5% to about 6% sorbitol. Ifsalt such as sodium chloride is added, then the effective range ofsucrose or sorbitol is relatively decreased. These and other suchosmolality and osmolarity considerations are well within the skill ofthe art.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or more freeradical oxidation inhibitors and/or chelating agents. A variety of freeradical scavengers and chelators are known in the art and apply to theformulations and methods of use described herein. Examples include butare not limited to ethanol, EDTA, a EDTA/ethanol combination,triethanolamine, mannitol, histidine, glycerol, sodium citrate, inositolhexaphosphate, tripolyphosphate, ascorbic acid/ascorbate, succinicacid/succinate, malic acid/maleate, desferal, EDDHA and DTPA, andvarious combinations of two or more of the above. In certainembodiments, at least one non-reducing free radical scavenger may beadded at a concentration that effectively enhances long term stabilityof the formulation. One or more free radical oxidationinhibitors/chelators may also be added in various combinations, such asa scavenger and a divalent cation. The choice of chelator will determinewhether or not the addition of a scavenger is needed.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or morenon-ionic surfactants, including but not limited to polyoxyethylenesorbitan fatty acid esters, Polysorbate-80 (Tween 80), Polysorbate-60(Tween 60), Polysorbate-40 (Tween 40) and Polysorbate-20 (Tween 20),polyoxyethylene alkyl ethers, including but not limited to Brij 58, Brij35, as well as others such as Triton X-100; Triton X-114, NP40, Span 85and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121),with preferred components Polysorbate-80 at a concentration from about0.001% to about 2% (with up to about 0.25% being preferred) orPolysorbate-40 at a concentration from about 0.001% to 1% (with up toabout 0.5% being preferred).

In certain embodiments, a formulation of the invention comprises one ormore additional stabilizing agents suitable for parenteraladministration, e.g., a reducing agent comprising at least one thiol(-SH) group (e.g., cysteine, N-acetyl cysteine, reduced glutathione,sodium thioglycolate, thiosulfate, monothioglycerol, or mixturesthereof). Alternatively or optionally, preservative-containingimmunogenic composition formulations of the invention may be furtherstabilized by removing oxygen from storage containers, protecting theformulation from light (e.g., by using amber glass containers).

Preservative-containing immunogenic composition formulations of theinvention may comprise one or more pharmaceutically acceptable carriersor excipients, which includes any excipient that does not itself inducean immune response. Suitable excipients include but are not limited tomacromolecules such as proteins, saccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers,sucrose (Paoletti et al, 2001, Vaccine, 19:2118), trehalose, lactose andlipid aggregates (such as oil droplets or liposomes). Such carriers arewell known to the skilled artisan. Pharmaceutically acceptableexcipients are discussed, e.g., in Gennaro, 2000, Remington: The Scienceand Practice of Pharmacy, 20^(th) edition, ISBN:0683306472.

Compositions of the invention may be lyophilized or in aqueous form,i.e.

solutions or suspensions. Liquid formulations may advantageously beadministered directly from their packaged form and are thus ideal forinjection without the need for reconstitution in aqueous medium asotherwise required for lyophilized compositions of the invention.

Direct delivery of immunogenic compositions of the present invention toa subject may be accomplished by parenteral administration(intramuscularly, intraperitoneally, intradermally, subcutaneously,intravenously, or to the interstitial space of a tissue); or by rectal,oral, vaginal, topical, transdermal, intranasal, ocular, aural,pulmonary or other mucosal administration. In a preferred embodiment,parenteral administration is by intramuscular injection, e.g., to thethigh or upper arm of the subject. Injection may be via a needle (e.g.,a hypodermic needle), but needle free injection may alternatively beused. A typical intramuscular dose is 0.5mL. Compositions of theinvention may be prepared in various forms, e.g., for injection eitheras liquid solutions or suspensions. In certain embodiments, thecomposition may be prepared as a powder or spray for pulmonaryadministration, e.g., in an inhaler. In other embodiments, thecomposition may be prepared as a suppository or pessary, or for nasal,aural or ocular administration, e.g., as a spray, drops, gel or powder.

Optimal amounts of components for a particular immunogenic compositionmay be ascertained by standard studies involving observation ofappropriate immune responses in subjects. Following an initialvaccination, subjects can receive one or several booster immunizationsadequately spaced.

Packaging and Dosage Forms

Immunogenic compositions of the invention may be packaged in unit doseor multi-dose form (e.g. 2 doses, 4 doses, or more). For multi-doseforms, vials are typically but not necessarily preferred over pre-filledsyringes. Suitable multi-dose formats include but are not limited to: 2to 10 doses per container at 0.1 to 2 mL per dose. In certainembodiments, the dose is a 0.5 mL dose. See, e.g., International PatentApplication WO2007/127668, which is incorporated by reference herein.

Compositions may be presented in vials or other suitable storagecontainers, or may be presented in pre-filled delivery devices, e.g.,single or multiple component syringes, which may be supplied with orwithout needles. A syringe typically but need not necessarily contains asingle dose of the preservative-containing immunogenic composition ofthe invention, although multi-dose, pre-filled syringes are alsoenvisioned. Likewise, a vial may include a single dose but mayalternatively include multiple doses.

Effective dosage volumes can be routinely established, but a typicaldose of the composition for injection has a volume of 0.5 mL. In certainembodiments, the dose is formulated for administration to a humansubject. In certain embodiments, the dose is formulated foradministration to an adult, teen, adolescent, toddler or infant (i.e.,no more than one year old) human subject and may in preferredembodiments be administered by injection.

Liquid immunogenic compositions of the invention are also suitable forreconstituting other immunogenic compositions which are presented inlyophilized form. Where an immunogenic composition is to be used forsuch extemporaneous reconstitution, the invention provides a kit withtwo or more vials, two or more ready-filled syringes, or one or more ofeach, with the contents of the syringe being used to reconstitute thecontents of the vial prior to injection, or vice versa.

Alternatively, immunogenic compositions of the present invention may belyophilized and reconstituted, e.g., using one of a multitude of methodsfor freeze drying well known in the art to form dry, regular shaped(e.g., spherical) particles, such as micropellets or microspheres,having particle characteristics such as mean diameter sizes that may beselected and controlled by varying the exact methods used to preparethem. The immunogenic compositions may further comprise an adjuvantwhich may optionally be prepared with or contained in separate dry,regular shaped (e.g., spherical) particles such as micropellets ormicrospheres. In such embodiments, the present invention furtherprovides an immunogenic composition kit comprising a first componentthat includes a stabilized, dry immunogenic composition, optionallyfurther comprising one or more preservatives of the invention, and asecond component comprising a sterile, aqueous solution forreconstitution of the first component. In certain embodiments, theaqueous solution comprises one or more preservatives, and may optionallycomprise at least one adjuvant (see, e.g., WO2009/109550 (incorporatedherein by reference).

In yet another embodiment, a container of the multi-dose format isselected from one or more of the group consisting of, but not limitedto, general laboratory glassware, flasks, beakers, graduated cylinders,fermentors, bioreactors, tubings, pipes, bags, jars, vials, vialclosures (e.g., a rubber stopper, a screw on cap), ampoules, syringes,dual or multi-chamber syringes, syringe stoppers, syringe plungers,rubber closures, plastic closures, glass closures, cartridges anddisposable pens and the like. The container of the present invention isnot limited by material of manufacture, and includes materials such asglass, metals (e.g., steel, stainless steel, aluminum, etc.) andpolymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).In a particular embodiment, the container of the format is a 5 mL SchottType 1 glass vial with a butyl stopper. The skilled artisan willappreciate that the format set forth above is by no means an exhaustivelist, but merely serve as guidance to the artisan with respect to thevariety of formats available for the present invention. Additionalformats contemplated for use in the present invention may be found inpublished catalogues from laboratory equipment vendors and manufacturerssuch as United States Plastic Corp. (Lima, Ohio), VWR.

EXAMPLES Example 1 Experimental Procedures Serum Bactericidal Assay

Cynomolgus macaques (n=5/group) were immunized intramuscularly withrLP2086 or rP2086 (A+B) proteins adsorbed to AlPO₄. Cynomolgus macaquesare an example of non-human primates. Animals were vaccinated at weeks0, 4 and 24, and ORF2086-specific IgG and functional antibody titerswere determined at weeks 0, 4, 6 and 26. Serum ORF2086-specific IgGtiters were determined against rLP2086A and B.

Functional antibody titers were examined by serum bactericidal assay(SBA) against Neisseria meningitidis strains expressing either LP2086with sequences homologous or heterologous to those contained in thevaccine.

Serum bactericidal antibodies in macaques or rabbits immunized withORF2086 vaccine were determined using SBAs with human complement. Rabbitimmune sera or macaques immune sera were heat-inactivated to removeintrinsic complement activity and subsequently serially diluted 1:2 inDulbecco's PBS with Ca2+ and Mg2+ (D-PBS) in a 96-well microtiter plateto test for serum bactericidal activity against N. meningitidis strains.Bacteria used in the assay were grown in GC media supplemented withKellogg's supplement (GCK) and monitored by optical density at 650 nm.Bacteria were harvested for use in the assay at a final OD₆₅₀ of0.50-0.55, diluted in D-PBS and 1000-3000 CFU were added to the assaymixture with 20% human complement.

Human serum with no detectable bactericidal activity was used as theexogenous complement source. Complement sources were tested forsuitability against each individual test strain. A complement source wasused only if the number of bacteria surviving in controls without addedimmune sera was >75%. Ten unique complement sources were required toperform the SBAs described in this study.

After a 30 min incubation at 37° C. with 5% CO₂, D-PBS was added to thereaction mixture and aliquots transferred to microfilter plates filledwith 50% GCK media. The microfilter plates were filtered, incubatedovernight at 37° C. with 5% CO₂ and microcolonies were stained andquantified. The serum bactericidal titers were defined as theinterpolated reciprocal serum dilution that yielded a 50% reduction inCFU compared to the CFU in control wells without immune sera. The SBAtiter is defined as the reciprocal of the interpolated dilution of testserum that causes a 50% reduction in bacterial counts after a 30 minincubation at 37° C. Susceptibility to killing with ORF2086 immune serawas established if there was a 4-fold or greater rise in SBA titer forORF2086 immune sera compared to the corresponding pre-immune sera. Serathat were negative against the assay strain at the starting dilutionwere assigned a titer of one half the limit of detection for the assay(i.e. 4).

Example 2 Cloning and Expression of Non-Lipidated ORF2086 Variants

The mature P2086 amino acid sequence corresponding to residues 27-286from N. meningitidis strain M98250771 (A05) was originally derived fromPCR amplification from genomic DNA. The forward primer, with a sequenceof TGCCATATGAGCAGCGGAAGCGGAAG (SEQ ID NO: 22), annealed to the 5′sequence and contained an NdeI site for cloning. The reverse primer,with a sequence of CGGATCCCTACTGTTTGCCGGCGATGC (SEQ ID NO: 23), annealedto the 3′ end of the gene and contained a termination codon TAG followedby restriction site BamHI. The 799 bp amplified fragment was firstcloned into an intermediate vector PCR2.1 (Invitrogen, Carlesbac,Calif.) This plasmid was cleaved with NdeI and BamHI, and was ligatedinto expression vector pET9a (Novagen, Madison, Wis.) which had beencleaved with NdeI and BamHI. The resulting vector pLA100 (which includesSEQ ID NO: 54), expressed the mature Subfamily A05 P2086 from strainM98250771 without the N-terminal cysteine (see SEQ ID NO: 13 wherein theN-terminal Cys at position 1 is deleted or SEQ ID NO: 55) that would bepresent in the lipidated protein. BLR(DE3) E. coli host strain [F-ompThsdSB(rB-mB-) gal dcm Δ(srI-recA)306::Tn10 (TetR) (DE3)] (Novagen) wasused to obtain expression of fHBP.

The same cloning steps were used to prepare the B02, B03, B09, B22, B24,B44, A04, A12, and A22 N-terminal Cys-deleted variants. The N-terminalCys-containing variants were also prepared by this same method usingforward primers which also included the Cys codon (e.g. the first codonof SEQ ID NOs: 1-11). Based on the sequences provided herein, theskilled worker would be able to design forward and reverse primers foreach of these variants. For example, the following primers were used toamplify the B44 non-lipidated variant followed by cloning into pET9ausing NdeI and BlpI.

TABLE 1 SEQ ID N-terminal Cys Primer Sequence NO Included-Fwd5′ TTTCTTcccgggAAGGAGatatac 24 atatgTGCAGCAGCGGAGGCGGCGG 3′ Included-Rev5′ TTTCTTgctcagcaTTATTGC 25 TTGGCGGCAAGACCGAT 3′ Deleted-Fwd5′ TTTCTTcccgggAAGGAGatataca 26 tatgAGCAGCGGAGGCGGCGG 3′ Deleted-Rev5′ TTTCTTgctcagcaTTATTGC 27 TTGGCGGCAAGACCGAT 3′

Results

Non-lipidated plasmid constructs were strongly expressed, but thenon-lipidated protein variants were pyruvylated at the N-terminal Cysresidue. See Examples 8 and 9, which describes, for example, a methodfor expressing the constructs. To overcome this pyruvylation, theN-terminal Cys codon was deleted. See, for example, Example 10. Deletionof the N-terminal Cys, however, abrogated expression of the A22 and B22variants. See e.g., FIG. 4. The A05, B01, and B44 variants, however,were still expressed despite deletion of the N-terminal Cys residue.See, for example, SEQ ID NO: 13 (A05), wherein the N-terminal Cys atposition 1 is deleted, SEQ ID NO: 35 (B01 N-terminus), and SEQ ID NO:21(B44),wherein the N-terminal Cys at position 1 is deleted. See e.g.,FIG. 5. In addition, expression of the non-lipidated B09 variant was notaffected by deletion of the N-terminal Cys residue. See, for example,Example 4.

Example 3 Effect of Gly/Ser Stalk on Non-Lipidated Variant Expression

To determine why the A05, B01, and B44 variants were expressed in theabsence of the N-terminal Cys and the A22 and B22 variants were not, thesequences of these variants were aligned. The A05, B01, and B44 variantsall possess an extended series of 10 or 11 Gly and Ser residuesimmediately following the N-terminal Cys (i.e. Gly/Ser stalk). The A22and B22 variants, however, only had a Gly/Ser stalk consisting of 6 Glyand Ser residues. Accordingly, the Gly/Ser stalk of the A22 and B22variants was expanded by insertion of additional Gly and Ser residues.

Long Gly/Ser stalk variants were prepared by the methods described inExample 2 using forward primers that encode a Gly/Ser stalk with either10 or 11 Gly and Ser residues.

The N-terminal Cys-deleted, long Gly/Ser stalk (10-11 Gly/Ser residues)A22 and B22 variants showed increased expression over the N-terminalCys-deleted A22 and B22 short Gly/Ser stalk (6 Gly/Ser residues)variants. These expression levels, however, were still reduced comparedto the A05, B01, and B44 variant expression levels.

Example 4 Codon Optimization

Expression of the non-lipidated B09 variant was not affected by deletionof the N-terminal Cys residue (see SEQ ID NO: 18, wherein the cysteineat position 1 is deleted, or SEQ ID NO: 49). See, e.g., FIG. 6. Sequenceevaluation of the B09 variant demonstrated that the B09 variant has aGly/Ser stalk consisting of 6 Gly and Ser residues, similar to theGly/Ser stalk of the A22 and B22 variants. Indeed, the N-terminal tailsof the B09 and A22 variants are identical at the amino acid level. TheN-terminal tails of the B09 and A22 variants (SEQ ID NO: 53 and 42,respectively), however, vary at the nucleic acid level by 2 nucleicacids: nucleic acids 15 and 39 of SEQ ID NO: 8. See e.g., FIG. 6. Thefirst 14 amino acids of the N-terminal tail of the B22 variant areidentical to the B09 and A22 variants, and the N-terminal tail of theB22 variant only differs at the 15th amino acid. Nucleic acids 1-42 ofthe B22 variant are identical to nucleic acids 1-42 of the A22 variant.Nucleic acids 1-42 of the B22 variant (see SEQ ID NO: 52) are identicalto nucleic acids 1-42 of B09 (see SEQ ID NO: 53) but for differences atnucleic acids 15 and 39, when optimally aligned. Accordingly, the B22variant differs from the B09 variant at amino acids 15 and 39 of SEQ IDNO: 8. This last sentence contains a typographical error and shouldstate that the B22 variant differs from the B09 variant at nucleic acids15 and 39 of SEQ ID NO: 8.

To determine if the nucleic acid differences affected the expressionlevel of the B09 variant compared to the A22 and B22 variants, the A22and B22 variants were mutated by point mutation to incorporate nucleicacids 15 and 39 into the corresponding codons for Gly5 and Glyl3.Incorporation of these silent nucleic acid mutations significantlyincreased expression of the A22 and B22 N-terminal Cys-deleted variantsto levels similar to the N-terminal Cys-deleted B09 variant. See e.g.,FIG. 7. Accordingly, codon optimization to match the B09 variant canincrease expression of N-terminal Cys-deleted non-lipidated P2086variants.

Further analysis of the non-lipidated variant sequences suggestedadditional codon optimizations in the Gly/Ser stalk to improveexpression. Accordingly, additional non-lipidated variants wereconstructed by the method of Example 2 using forward primers comprisingsuch codon optimized sequences. The forward primers used to generateoptimized Gly/Ser stalks include any of the following sequences:

(SEQ ID NO: 28) ATGAGCTCTGGAGGTGGAGGAAGCGGGGGCGGTGGA (SEQ ID NO: 29)  M  S  S   G  G  G   G  S  G  G  G  G (SEQ ID NO: 30)ATGAGCTCTGGAAGCGGAAGCGGGGGCGGTGGA (SEQ ID NO: 31)  M  S   S  G  S   G  S  G  G  G  G  (SEQ ID NO: 32)ATGAGCTCTGGAGGTGGAGGA (SEQ ID NO: 33)   M  S   S  G  G  G   G(SEQ ID NO: 34) ATGAGCAGCGGGGGCGGTGGA (SEQ ID NO: 33)  M   S  S  G  G  G  G

Example 5 Immunogenic Composition Formulation Optimization

ISCOMATRIX formulated vaccines generate a rapid immune responseresulting in a reduction in the number of dosages required to achieve agreater than 4 fold response rate as measured in a serum bactericidalassay. Groups of five rhesus macaques were immunized with differentformulations of a bivalent non-lipidated rP2086 vaccine. The vaccineincluded a non-pyruvylated non-lipidated A05 variant (SEQ ID NO: 13wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 55encoded by SEQ ID NO: 54) and a non-pyruvylated non-lipidated B44variant (SEQ ID NO: 21 wherein the N-terminal Cys at position 1 isdeleted or SEQ ID NO: 44 encoded by SEQ ID NO: 51). The adjuvant unitsare as follows: AlPO₄ is 250 mcg, ISCOMATRIX is between 10 and 100 mcg.The adjuvant units for AlPO₄ shown in Tables 2-5 are shown as milligramunits, and are therefore shown as 0.25 (milligram) as opposed to 250mcg.

The immunization schedule was 0, 4 and 24 wks with bleeds at 0, 4, 6 and26 weeks. There were no increases in SBA titers at post dose one for anyof the groups. At post dose two, an increase in SBA titers and thenumber of responders as defined by a 4 fold increase in SBA titer abovebaseline was observed for formulations containing the ISCOMATRIXadjuvant. Tables 2 and 3 provide the SBA GMTs observed for a fHBPSubfamily A and B strain respectively. SBA GMTs for the ISCOMATRIXformulations were 3-19 and 4-2 4 fold higher than those observed for theAlPO4 formulation for the A and B subfamily strains respectively.Enhanced titers were also observed at post dose three for the ISCOMATRIXformulations at 13-95 and 2-10 for a fHBP Subfamily A and B strainrespectively compared to the AlPO4 formulation. Analysis of theresponder rates, as defined by a four fold or greater increase in SBAtiter over baseline revealed a similar trend (Tables 4 and 5).

TABLE 2 SBA titers (GMTs) obtained for against a MnB LP2086 Subfamily Astrain immune serum from rhesus macaques immunized with differentformulations of a bivalent rP2086 vaccine Adjuvant Geometric Mean titer(GMT) Vaccine lipidation AlPO4 ISCOMATRIX ® wk 0 wk 4 wk 6 wk 26 A05/B44− 0.25 − − − − + − 10 − − + +++ 0.25 10 − − + ++ − 100 − − ++ ++++ 0.25100 − − + +++ Five monkeys per group; Immunization schedule: 0, 4, 24weeks; bleed schedule 0, 4, 6 and 26 wks. SBA test strain MnB M98250771. “−” <8; “+” 8-32; “++” 33-128; “+++” 129-512; “++++” >512

TABLE 3 SBA titers (GMTs) obtained for against a MnB LP2086 Subfamily Bstrain immune serum from rhesus macaques immunized with differentformulations of a bivalent rP2086 vaccine Adjuvant Geometric Mean titer(GMT) Vaccine lipidation AlPO4 ISCOMATRIX ® wk 0 wk 4 wk 6 wk 26 A05/B44− 0.25 − − − + +++ − 10 − − +++ ++++ 0.25 10 − − +++ ++++ − 100 − − +++++++ 0.25 100 − − ++ ++++ Five monkeys per group; Immunization schedule:0, 4, 24 weeks; bleed schedule 0, 4, 6 and 26 wks. SBA test strain MnBCDC1127. “−” <8; “+” 8-32; “++” 33-128; “+++” 129-512; “++++” >512

TABLE 4 Number of rhesus macaques with a ≥4 fold rise in SBA Titer usinga MnB LP2086 Subfamily A strain Adjuvant No. of responders^(b) Vaccinelipidation AlPO4 ISCOMATRIX ® wk 0 wk 4 wk 6 wk 26 A05/B44 − 0.25 − 0 00 2 − 10 0 0 3 5 0.25 10 0 0 2 5 − 100 0 0 4 5 0.25 100 0 0 2 5

TABLE 5 Number of rhesus macaques with a ≥4 fold rise in SBA Titer usinga MnB LP2086 Subfamily B strain Adjuvant No. of responders^(b) Vaccinelipidation AlPO4 ISCOMATRIX ® wk 0 wk 4 wk 6 wk 26 A05/B44 − 0.25 − 0 03 5 − 10 0 0 5 5 0.25 10 0 0 5 5 − 100 0 0 4 4 0.25 100 0 0 3 5

Example 6 Immunoprotection Conferred by Lipidated and Non-LipidatedVariants

A recombinantly expressed non-lipidated P2086 variant (B44) inducesbroad protection as measured by SBA against strains that representdiverse fHBP variants (from about 85% to about <92% ID) LP2086sequences. These response rates were obtained for a non lipidatedvaccine formulated with A lPO₄. See Table 6, which shows SBA responserates to a subfamily B fHBP MnB strain generated by a bivalent fHBPvaccine. The non-lipidated vaccine (represented by a “² under the“lipidation” column) included 1 mcg per protein of a non-pyruvylatednon-lipidated A05 variant (SEQ ID NO: 13 wherein the N-terminal Cys atposition 1 is deleted) and a non-pyruvylated non-lipidated B44 variant(SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted).

Alternatively, a recombinantly expressed non-lipidated P2086 variant(B44) induces greater immune responses as measured by SBA titer than alipidated variant (B01) against strains bearing similar (>92% ID) anddiverse (<92% ID) LP2086 sequences. Higher response rates (as defined bya four fold increase or greater in SBA titers over baseline) wasobserved for the vaccine containing the non-lipidated rP2086 B44compared to the lipidated rLP2086 B01 vaccine (Table 6).

According to Table 6, non-lipidated B44 is a preferred subfamily Bcomponent of fHBP in a composition for providing broad coverage against(e.g., eliciting bactericidal antibodies against) multiple LP2086variant strains.

Surprisingly, the inventors noted that LP2086 B09 variant strains areparticularly unlikely to have positive SBA response rates with regard toheterologous (non-B09) ORF2086 polypeptides. In particular, theinventors found that LP2086 B09 is an exception in terms of an assaystrain against which the A05/B44 immunogenic composition described inTable 6 elicited bactericidal antibodies. Therefore, in a preferredembodiment an immunogenic composition of the invention includes a B09polypeptide, in particular in the context of a composition includingmore than one ORF2086 subfamily B polypeptide. In a preferred embodimentan immunogenic composition that includes a non lipidated B44 may alsoinclude a non-lipidated B09 polypeptide.

TABLE 6 SBA response rates to a Subfamily B fHBP MnB strains generatedby bivalent fHBP vaccines Immune serum from rhesus macaques. % ID toMatched Subfamilyfor non- LP2086 Variant lipidated Vaccine % respondersAdjuvant of Assay Strain Vaccine lipidation Component PD3 Wk 26 B02A05/B01 + 99.6 80 A05/B44 − 100 AlPO4 B03 A05/B01 + 86.7 50 0.25 mgA05/B44 − 80 B09 A05/B01 + 86.3 0 A05/B44 − 0 B15 A05/B01 + 86.7 25A05/B44 − 80 B16 A05/B01 + 87.1 0 A05/B44 − 50 B16 A05/B01 + 87.1 0A05/B44 − 60 B24 A05/B01 + 85.9 0 A05/B44 − 60 B44 A05/B01 + 100 100A05/B44 − 100 ISCOMATRIX ® A05 A05/B44 − 100 100 (10 mcg) ISCOMATRIX ®A05 A05/B44 − 100 100 (100 mcg) ISCOMATRIX ® A22 A05/B44 − 88.9 80 (10mcg) ISCOMATRIX ® A22 A05/B44 − 88.9 100 (100 mcg) Five monkeys pergroup; Immunization schedule: 0, 4, 24 weeks; bleed schedule 0, 4, 6,and 26 wks.

Example 7 Codon Optimization of the B44 and B09 Variants

Although the expression levels achieved in the preceding examples wereadequate for many applications, further optimization was desirable, andE. coli expression constructs containing additional codon optimizationover the full length of the protein were prepared and tested. One suchimproved sequence for expression of a non-Cys B44 protein was found tobe the nucleic acid sequence set forth in SEQ ID NO: 43. As shown inExample 9, the expression construct containing SEQ ID NO: 43 showedenhanced expression compared to that of the non-optimized wild typesequence.

Expression of the N-terminal Cys deleted B09 protein was improved byapplying codon changes from the above optimized B44 (SEQ ID NO: 43)construct to B09 (SEQ ID NO: 48). To generate optimized B09 sequences,the B44 optimized DNA sequence (SEQ ID NO: 43) was first aligned to theDNA sequence of the B09 allele (SEQ ID NO: 48). The entire non-lipidatedcoding sequence of the B09 allele (SEQ ID NO: 48) was optimized toreflect the codon changes seen in the B44 optimized allele (SEQ ID NO:43) wherever the amino acids between B44 (SEQ ID NO: 44) and B09 (SEQ IDNO: 49) were identical. Codon sequences in the B09 allele correspondingto the identical amino acids between the B09 allele and the B44 allelewere changed to reflect the codon used in the B44 optimized sequence(SEQ ID NO: 43). Codon sequences for amino acids that differ between B09(SEQ ID NO: 49) and B44 (SEQ ID NO: 44) were not changed in the B09 DNAsequence.

Additionally, the non-lipidated B44 amino acid sequence (SEQ ID NO: 44)contains two sequential serine-glycine repeat sequences (S-G-G-G-G) (SEQID NO: 56)(see also amino acids 2 to 6 of SEQ ID NO: 44) at itsN-terminus, whereas the B09 allele contains only one serine-glycinerepeat at the N-terminus (see amino acids 2 to 6 and amino acids 7 to 11of SEQ ID NO: 49). The two serine-glycine repeats at the N-terminus ofB44 (amino acids 2 to 6 and amino acids 7 to 11 of SEQ ID NO: 44) alsohave different codon usage (see nucleotides 4 to 18 and nucleotides 19to 33 of SEQ ID NO: 43), and different combinations of the optimized B44serine-glycine repeat (e.g., either nucleotides 4 to 18 of SEQ ID NO:43, or nucleotides 19 to 33 of SEQ ID NO: 43, or a combination thereof)were applied to the B09 DNA sequence (SEQ ID NO: 48, e.g., applied tonucleotides 4 to 18 of SEQ ID NO: 48) in order to examine the effect onrecombinant protein expression.

Three different versions of optimized B09 were constructed: SEQ ID NO:45 contains both serine-glycine repeats (GS1 and GS2) (nucleic acids 4to 33 of SEQ ID NO: 43) from the optimized B44, SEQ ID NO: 46 containsGS1 (nucleic acids 4 to 18 of SEQ ID NO: 43), and SEQ ID NO: 47 containsGS2 (nucleic acids 19 to 33 of SEQ ID NO: 43). The DNA for all of theabove codon optimized sequences were chemically synthesized usingstandard in the art chemistry. The resulting DNA was cloned intoappropriate plasmid expression vectors and tested for expression in E.coli host cells as described in Examples 8 and 9.

Example 8 Method for Expressing ORF2086, B09 Variant

Cells of E. coli K-12 strain (derivatives of wild-type W3110 (CGSC4474)having deletions in recA, fhuA and araA) were transformed with plasmidpEB063, which includes SEQ ID NO: 45, pEB064, which includes SEQ ID NO:46, plasmid pEB065, which includes SEQ ID NO: 47, or plasmid pLA134,which includes SEQ ID NO: 48. The preferred modifications to the K-12strain are helpful for fermentation purposes but are not required forexpression of the proteins.

Cells were inoculated to a glucose-salts defined medium. After 8 hoursof incubation at 37° C. a linear glucose feed was applied and incubationwas continued for an additional 3 hours. Isopropyl6-D-1-thiogalactopyranoside (IPTG) was added to the culture to a finalconcentration of 0.1 mM followed by 12 hours of incubation at 37° C.Cells were collected by centrifugation at 16,000×g for 10 minutes andlysed by addition of Easy-Lyse™ Cell Lysing Kit” from LiencoTechnologies (St. Louis, Mo.) and loading buffer. The cleared lysateswere analyzed for expression of B09 by Coomassie staining of SDS-PAGEgels and/or Western blot analysis with quantitation by a scanningdensitometer. The results from scanning densitometry are below in Table7:

TABLE 7 Expression data in E. coli Percentage of total cell protein at12 hours post IPTG induction, as measured by SDS-PAGE, scanning ProteinHost cell Plasmid desitometry B09 E. coli K-12 pEB063 24% SEQ ID NO: 45B09 E. coli K-12 pEB065 12% SEQ ID NO: 47 B09 E. coli K-12 pEB064 38%SEQ ID NO: 46 B09 E. coli K-12 pLA134 13% SEQ ID NO: 48

Example 9 Method for Expressing ORF2086, B44 Variant

Cells of E. coli B strain (BLR(DE3), Novagen) were transformed withplasmid pLN056, which includes SEQ ID NO: 51. Cells of E. coli K-12strain (derivative of wild-type W3110) were transformed with plasmidpDK087, which includes SEQ ID NO: 43. Cells were inoculated to aglucose-salts defined medium. After 8 hours of incubation at 37° C. alinear glucose feed was applied and incubation was continued for anadditional 3 hours. Isopropyl β-D-1-thiogalactopyranoside (IPTG) wasadded to the culture to a final concentration of 0.1 mM followed by 12hours of incubation at 37° C. Cells were collected by centrifugation at16,000×g for 10 minutes and lysed by addition of Easy-Lyse™ Cell LysingKit” from Lienco Technologies (St. Louis, Mo.) and loading buffer. Thesupermatants were analyzed for expression of B09 by Coomassie stainingof SDS-PAGE gels and/or Western blot analysis, with quantitation by ascanning densitometer. The results from scanning densitometry are belowin Table 8:

TABLE 8 Expression data in E. coli Percentage of total cell protein at12 hours post IPTG induction, as measured by SDS-PAGE, scanning ProteinHost cell Plasmid desitometry. B44 E. coli B pLN056  1% SEQ ID NO: 51B44 E. coli K-12 pDK087 17% SEQ ID NO: 43

Example 10 Pyruvylation

The present example demonstrates that the N-terminal Cys residue ofnon-lipidated ORF2086 proteins can become pyruvylated when expressed in,for example, E. coli.

Heterologous protein accumulation during production of variants A05 (SEQID NO: 13) and B44 (SEQ ID NO: 21) were monitored using reverse-phasehigh performance liquid chromatography (RP-HPLC). This separation wasinterfaced with a quadrupole time-of-flight mass spectrometer (QTOF-MS)to provide a means of monitoring formation of product related variants.

After being expressed in the E. coli B and/or K-12 host cells, productsderived from these fermentations underwent a purification procedureduring which a product modification was observed. Deconvolution of themass spectra characterized the variants as exhibiting mass shifts of +70Da, as compared to native products of 27640 and 27572 Da for A05 andB44, respectively.

Published literature indicated that a +70 Da mass shift had previouslybeen observed in proteins and has been attributed to pyruvylation of theamino-terminal residue.

The presence and location of the pyruvate group was confirmed using themass spectral fragmentation data (MS/MS). The data indicated that themodification was on an amino-terminal cysteine residue, i.e., amino acidat position 1, according to A05 and B44. For A05, the percentage ofpyruvylated polypeptides was about 30%, as compared to the total numberof A05 polypeptides (SEQ ID NO: 13). For B44 the percentage ofpyruvylated polypeptides was about 25%, as compared to the total numberof B44 polypeptides (SEQ ID NO: 21).

When A05 (SEQ ID NO: 13 wherein the N-terminal Cys at position 1 isdeleted or SEQ ID NO: 55) and B44 variants (SEQ ID NO: 21 wherein theN-terminal Cys at position 1 is deleted or SEQ ID NO: 44), which do notcontain an amino-terminal cysteine, were purified, there was nodetectable pyruvylation (+70 Da).

Example 11 Immunogenicity of B09 and B44, Individually and inCombination

5 -10 groups of rhesus maccaques monkeys were immunized with B09 variant(SEQ ID NO: 49 encoded by SEQ ID NO: 48) or B44 variant (SEQ ID NO: 44encoded by SEQ ID NO: 43), or the A05, B09 and B44 (SEQ ID NO: 55, SEQID NO: 49 encoded by SEQ ID NO: 48, and SEQ ID NO: 44 encoded by SEQ IDNO: 43, respectively) formulated with 250 mcg of AlPO₄ per dose. Themonkeys were vaccinated via the intramuscular route at weeks 0, 4 and 8with 10 mcg each of non-lipidated fHBP alone or in combination as listedin Table 9 and 10. Both weeks 0 and 12 serum samples were analyzed inSBAs against MnB strains with either subfamily A or subfamily B fHBPvariants. Responders were recorded as animals with a 4× rise in titer.The B44 variant tested was the optimized construct (SEQ ID NO: 43) andthe broad response rates that were observed in previous studies (tableabove) were maintained for the optimized construct (Table 9) the B44vaccine alone or in combination with B09. The B09 vaccine alone (Table10) could also generate broadly cross reactive immune responses (Table10).

TABLE 9 Response rates obtained for non lipidated fHBP vaccines inrhesus macaques % ≥ 4 X Rise Against Test Variant (PD3; 10 rhesusmacaques per group) Vaccine A05 B44 B16 B24 B09 (10 mcg (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID per protein; NO: 13) NO: 21) NO: 60) NO: 20) NO:18) B44 0 80 30 40 30 B44 + B09 + 60 80 40 50 30 A05

-   Rhesus macaques (n=10) were immunized i.m. at weeks 0, 4 and 8 with    10 mcg each of non-lipidated fHBP alone or in combination as listed    in the Vaccine column in formulation with 250 mcg of AlPO₄. Both    weeks 0 and 10 serum samples were analyzed in SBAs against the MnB    strains listed in the table. Responders are recorded as animals with    a 4× rise in titer.

Table 9 indicates, for example, that a composition including acombination of non-pyruvylated non-lipidated B44, B09, and A05 showedhigher cross-coverage against the test variants as compared to thecross-coverage from a composition including B44 alone. In view ofresults shown in the present application, including in particular Table6 and Table 9 together, compositions including B44, B09 and A05 alone orin combination are preferred embodiments of the present invention. Inparticular, compositions including both B44 and B09 are disclosed. Suchcomposition preferably further includes a subfamily A polypeptide, suchas in particular A05.

TABLE 10 Response rates obtained for non lipidated fHBP B09 vaccine inrhesus macaques % ≥ 4 X Rise Against Test Variant Vaccine (10 mcg (PD3;5 rhesus macaques per group) per protein) A05 B44 B16 B24 B09 B09 40 6040 60 60

-   Rhesus macaques (n=5) were immunized i.m. at weeks 0, 4 and 8 with    10 mcg each of non-lipidated fHBP alone or in combination as listed    in the Vaccine column in formulation with 250 mcg of AlPO₄. Both    weeks 0 and 10 serum samples were analyzed in SBAs against the MnB    strains listed in the table. Responders are recorded as animals with    a 4× rise in titer.

Example 12 Immunoprotection Conferred by Lipidated and Non-LipidatedVariants Construct

Twenty female New Zealand white rabbits, 2.5-3.5 kg, obtained fromCharles River Canada, were pre-screened by whole cell ELISA and 10animals were selected for this study based on their low backgroundtiters against the test strains representing fHBP variants B02 (SEQ IDNO: 16) and B44 (SEQ ID NO: 21) (Table 11). Group of three animals werei.m. immunized with 100 μg of each protein formulated with 50 μgISCOMATRIX per 0.5 ml dose at weeks 0, 4 and 9 (Table 12). Group 1 wasvaccinated with non-lipidated B44 (SEQ ID NO: 44). A control group wasincluded that was vaccinated with lipidated B01 formulated with A lPO4(250 mcg) Rabbits were bled at weeks 0, 4, 9 and 10. Individual serafrom week 10 were prepared and analyzed by serum bactericidal assayagainst multiple serogroup B meningococcal strains from the fHBP Bsubfamily.

TABLE 11 Rabbits Used in The Study Species: Rabbit Strain: New Zealandwhite Source:^(a) Charles River Laboratory No. of Animals Per Group: 3Total No. of Animals: 9 Age and Sex: Female Weight: 2.5-3.5 kg

TABLE 12 Aluminium rfHBP ISCOMATRIX Phosphate # of (μg/0.5 ml (μg/0.5 ml(μg/0.5 ml Group animals Variant lipidated dose) dose) dose) 1 3 B44 −100 50 2 3 B01 − 100 50 3 3 B01 + 100 − 100

Immunization Schedule Weeks 0, 4, 9; Bleed Schedule Weeks 0, 4, 9,10

-   Serum Bactericidal Assay (SBA): A microcolony-based serum    bactericidal assay (SBA) against multiple serogroup B meningococcal    strains (Table 13) was performed on individual serum samples. Human    sera from donors were qualified as the complement source for the    strain tested in the assay. Complement-mediated antibody-dependent    bactericidal titers were interpolated and expressed as the    reciprocal of the dilution of the test serum that killed 50% of the    meningococcal cells in the assay. The limit of detection of the    assay was an SBA titer of 4. An SBA titer of <4 was assigned number    of 2. A ≥4-fold rise of SBA titers in the week 10 sera in comparison    to the titers in the pre-bleed was calculated and compared.

Serum bactericidal antibody activity as measured in the SBA is theimmunologic surrogate of protection against meningococcal disease. Theability of immunization with non-lipidated rfHBP to elicit bactericidalantibodies in rabbits was determined by SBA. SBA measures the level ofantibodies in a serum sample by mimicking the complement-mediatedbacterial lysis that occurs naturally. Rabbit serum samples collectedfrom week 10 were analyzed by SBA against strains with a B44 fHBP or aB02 fHBP. As shown in Table 13, one week after the third immunization(week 10), all serum samples displayed bactericidal activity againstboth test strains. (Table 13). The non-lipidated B44 (SEQ ID NO: 44) wasmore immunogenic than non-lipidated B01 in New Zealand Rabbits againstthese strains. The non lipidated B44 (SEQ ID NO: 44) formulated with theiscomatrix adjuvant gave comparable titers to the lipidated B01formulated with aluminium phosphate against these strains. Rabbitpre-bleed sera showed generally no pre-existing bactericidal activityagainst the tested strains.

TABLE 13 Serum Bactericidal Activity against fHBP Subfamily B Strains inNew Zealand White Rabbits Vaccinated with Recombinant Non-lipidated fHBPGMT SBA Titer against test variant Subfamily B variant B44 (SEQ B02 (SEQ(formulation) ID NO: 21) ID NO: 16) Non lipidated B44 (SEQ ID 6675 7140NO: 44)(Iscomatrix) Non lipidated B01 625 1052 (ISCOMATRIX) LipidatedB01 (AlPO₄) 10099 10558

Example 13 Immunogenicity of Six Non-Lipidated Factor H Binding Proteinsin New Zealand White Rabbits

Groups of 5 rabbits were immunized with non-lipidated fHBP variants asdescribed in Table 14. Vaccines were administered at 0, 4 and 9 weeks.Rabbit serum samples collected from weeks 0 and 10 were analyzed by SBAagainst the strains with homologous and heterologous fHBP sequences.Table 14 shows the percent responders post the third immunization. Oneweek after the third immunization (week 10), all serum samples displayedbactericidal activity against the homologous strains as well as othertest strains from the same fHBP subfamily. Rabbits pre-bleed sera showedgenerally no pre-existing bactericidal activity against the testedstrains.

TABLE 14 Post Dose Three Percent of Responders in New Zealand WhiteRabbits Vaccinated with Recombinant Non-lipidated fHBPs MnB Dose/0.5fHBP mL AlPO4/0.5 mL n B09 B16 B24 B44 A05 A12 A22 A05 100 mcg 0.25 mg 5100 80 100 A12 100 mcg 0.25 mg 5 100 100 100 A22 100 mcg 0.25 mg 5 80 8080 B09 100 mcg 0.25 mg 5 100 80 60 80 B22 100 mcg 0.25 mg 5 40 100 60100 B44 100 mcg 0.25 mg 5 0 60 40 100 A05, 100 mcg 0.25 mg 5 100 100 60100 100 100 100 A12, each/400 B22, mcg total B44

A05 SEQ ID NO: 13, wherein the Cys at position 1 is deleted, or SEQ IDNO: 55 encoded by SEQ ID NO: 54 A12 SEQ ID NO: 14, wherein the Cys atposition 1 is deleted A22 SEQ ID NO: 15, wherein the Cys at position 1is deleted B09 SEQ ID NO: 18, wherein the Cys at position 1 is deleted,or SEQ ID NO: 49 encoded by SEQ ID NO: 48. B22 SEQ ID NO: 19, whereinthe Cys at position 1 is deleted B44 SEQ ID NO: 21, wherein the Cys atposition 1 is deleted, or SEQ ID NO: 44 encoded by SEQ ID NO: 51

Test variants in Table 14: B09 B16 B24 B44 A05 A12 A22 (SEQ ID (SEQ (SEQID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 18) ID NO: 60) NO: 20) NO: 21)NO: 13) NO: 14) NO: 15)The invention also provides the following embodiments as defined in theclauses below:

C1. An immunogenic composition comprising a P2086 polypeptide, whereinthe P2086 is a B44, a B02, a B03, a B22, a B24, a B09, an A05, an A04,an A12, or an A22 variant.

C2. An immunogenic composition comprising a P2086 Subfamily Bpolypeptide, wherein the P2086 Subfamily B polypeptide is a B44, a B02,a B03, a B22, a B24 or a B09 variant.

C3. The immunogenic composition of C2 further comprising a P2086Subfamily A polypeptide.

C4. The immunogenic composition of C3, wherein the P2086 Subfamily Apolypeptide is an A05, an A04, an A12, or an A22 variant.

C5. The immunogenic composition of any one of 01-4, wherein thecomposition further comprises an adjuvant.

C6. The immunogenic composition of C5, wherein the adjuvant is selectedfrom the group consisting of:

-   -   a) an aluminum adjuvant;    -   b) a saponin    -   c) a CpG nucleotide sequence; and    -   d) any combination of a), b) and c).

C7. The immunogenic composition according to C6, wherein the aluminumadjuvant is selected from the group consisting of AlPO₄, Al(OH)₃,Al₂(SO₄)₃ and alum.

C8. The immunogenic composition according to C6 or C7, wherein theconcentration of aluminum is between 0.125 μg/ml and 0.5 μg/ml.

C9. The immunogenic composition according to C8, wherein theconcentration of aluminum is 0.25 μg/ml.

C10. The immunogenic composition according to any one of C6-9, whereinthe saponin concentration is between 1 μg/ml and 250 μg/ml.

C11. The immunogenic composition according to C10, wherein the saponinconcentration is between 10 μg/ml and 100 μg/ml.

C12. The immunogenic composition according to C10, wherein the saponinconcentration is 10 μg/ml.

C13. The immunogenic composition according to C10, wherein the saponinconcentration is 100 μg/ml.

C14. The immunogenic composition according to any one of C6-13, whereinthe saponin is QS-21 or ISCOMATRIX.

C15. The immunogenic composition according to any one of C1-14, whereinthe composition confers the ability to raise an immunogenic response toa Neisseria meningitidis bacteria after administration of multiple dosesto a subject.

C16. The immunogenic composition according to C15, wherein theimmunogenic response to the Neisseria meningitidis bacteria is conferredafter administration of 2 doses to the subject.

C17. The immunogenic composition according to C15, wherein theimmunogenic response to the Neisseria meningitidis bacteria is conferredafter administration of 3 doses to the subject.

C18. A composition conferring increased immunogenicity on anon-lipidated P2086 antigen, wherein the composition comprises a saponinand at least one non-lipidated P2086 antigen.

C19. The immunogenic composition according to C18, wherein the saponinconcentration is between 1 μg/ml and 250 μg/ml.

C20. The immunogenic composition according to C19, wherein the saponinconcentration is between 10 μg/ml and 100 μg/ml.

C21. The immunogenic composition according to C19, wherein the saponinconcentration is 10 μg/ml.

C22. The immunogenic composition according to C19, wherein the saponinconcentration is 100 μg/ml.

C23. The immunogenic composition according to any one of C18-22, whereinthe saponin is QS-21 or ISCOMATRIX.

C24. The immunogenic composition according to any one of C18-23 furthercomprising aluminum.

C25. The immunogenic composition according to C24, wherein theconcentration aluminum is between 0.125 μg/ml and 0.5 μg/ml.

C26. The immunogenic composition according to C25, wherein theconcentration of aluminum is 0.25 μg/ml.

C27. The immunogenic composition according to any one of C18-26, whereinthe composition confers an immunogenic response to a Neisseriameningitidis bacteria after administration of multiple doses to thesubject.

C28. The immunogenic composition according to C27, wherein theimmunogenic response to the Neisseria meningitidis bacteria is conferredafter administration of 2 doses to the subject.

C29. The immunogenic composition according to C27, wherein theimmunogenic response to the Neisseria meningitidis bacteria is conferredafter administration of 3 doses to the subject.

C30. The immunogenic composition according any one of C18-29, whereinthe non-lipidated P2086 antigen is a non-lipidated P2086 Subfamily Bpolypeptide.

C31. The immunogenic composition according to C30, wherein thenon-lipidated P2086 Subfamily B polypeptide is a B44, a B02, a B03, aB22, a B24 or a B09 variant.

C32. The immunogenic composition according any one of C18-29, whereinthe non-lipidated P2086 antigen is a non-lipidated P2086 Subfamily Apolypeptide.

C33. The immunogenic composition according to C32, wherein thenon-lipidated P2086 Subfamily A polypeptide is an A05, an A04, an A12,or an A22 variant.

C34. The immunogenic composition according any one of C18-33, whereinthe composition comprises at least two non-lipidated P2086 antigens,wherein the two non-lipidated P2086 antigens are at least onenon-lipidated P2086 Subfamily A polypeptide and at least onenon-lipidated P2086 Subfamily B polypeptide.

C35. The immunogenic composition according to C34, wherein thenon-lipidated P2086 Subfamily A polypeptide is an A05 variant and thenon-lipidated P2086 Subfamily B polypeptide is a B44 variant.

C36. The immunogenic composition according to C34, wherein thenon-lipidated P2086 Subfamily A polypeptide is an A05 variant and thenon-lipidated P2086 Subfamily B polypeptide is a B22 variant.

C37. The immunogenic composition according to C34, wherein thenon-lipidated P2086 Subfamily A polypeptide is an A05 variant and thenon-lipidated P2086 Subfamily B polypeptide is a B09 variant.

C38. A method for conferring immunity to a subject against a Neisseriameningitidis bacteria, wherein the method comprises the step ofadministering to the subject an immunogenic composition comprising aP2086 Subfamily B polypeptide, wherein the P2086 Subfamily B polypeptideis a B44, a B02, a B03, a B22, a B24 or a B09 variant.

C39. The method according to C38, wherein the immunogenic compositionfurther comprises an a P2086 Subfamily A polypeptide.

C40. The method according to C39, wherein the P2086 Subfamily Apolypeptide is an A05, an A04, an A12, or an A22 variant.

C41. The method according to any one of C38-40, wherein the immunogeniccomposition further comprises an adjuvant.

C42. The method according to C41, wherein the adjuvant is selected fromthe group consisting of:

-   -   a) an aluminum adjuvant;    -   b) a saponin    -   c) a CpG nucleotide sequence; and    -   d) any combination of a), b) and c).

C43. The method according to C42, wherein the aluminum adjuvant isselected from the group consisting of AlPO₄, Al(OH)₃, Al₂(SO₄)₃ andalum.

C44. The method according to C42 or 43, wherein the concentration ofaluminum is between 0.125 μg/ml and 0.5 μg/ml.

C45. The method according to C44, wherein the concentration of aluminumis 0.25 μg/ml.

C46. The method according to any one of C42-45, wherein the saponinconcentration is between 1 μg/ml and 250 μg/ml.

C47. The method according to C46, wherein the saponin concentration isbetween 10 μg/ml and 100 μg/ml.

C48. The method according to C47, wherein the saponin concentration is10 μg/ml.

C49. The method according to C48, wherein the saponin concentration is100 μg/ml.

C50. The method according to any one of C42-49, wherein the saponin isQS-21 or ISCOMATRIX.

C51. The method according to C38-50, wherein the immunogenic compositionis administered to the subject in multiple doses over a dosing schedule.

C52. The method according to C51, wherein the immunogenic composition isadministered to the subject in 2 doses over a dosing schedule.

C53. The method according to C51, wherein the immunogenic composition isadministered to the subject in 3 doses over a dosing schedule.

C54. A method for producing a non-lipidated P2086 variant polypeptidecomprising the steps of:

-   -   a) cloning the ORF2086 variant nucleic acid sequence into an E.        coli expression vector;    -   b) transforming bacteria with the ORF2086 expression vector;    -   c) inducing expression; and    -   d) isolating the expressed P2086 protein;        wherein, the ORF2086 expression vector does not comprise a        lipidation control sequence.

C55. The method according to C54, wherein the codon encoding theN-terminal Cys of the ORF2086 variant is deleted.

C56. The method according to C54, wherein the codon encoding theN-terminal Cys of the ORF2086 variant is mutated to generate an Ala,Gly, or Val codon.

C57. The method according to C55 or 56, wherein the ORF2086 variant isan A05, a B01, or a B44 variant.

C58. The method according to any one of C54-57, wherein the N-terminaltail is mutated to add Ser and Gly residues to extend the Gly/Ser stalkimmediately downstream of the N-terminal Cys.

C59. The method according to C58, wherein the total number of Gly andSer residues in the Gly/Ser stalk is at least 7.

C60. The method according to C58, wherein the total number of Gly andSer residues in the Gly/Ser stalk is at least 8.

C61. The method according to C58, wherein the total number of Gly andSer residues in the Gly/Ser stalk is at least 9.

C62. The method according to C58, wherein the total number of Gly andSer residues in the Gly/Ser stalk is at least 10.

C63. The method according to C58, wherein the total number of Gly andSer residues in the Gly/Ser stalk is at least 11.

C64. The method according to C58, wherein the total number of Gly andSer residues in the Gly/Ser stalk is at least 12.

C65. The method according to any one of C54-57, wherein the codons ofthe N-terminal tail of the ORF2086 variant are optimized by pointmutagenesis such that the codon encoding the fifth amino acid of theORF2086 variant is 100% identical to nucleotides 13-15 of SEQ ID NO: 8and the codon encoding the thirteenth amino acid of the ORF2086 variantis 100% identical to nucleotides 37-39 of SEQ ID NO: 8.

C66. The method according to C65, wherein the codons of the N-terminaltail of the ORF2086 variant are 100% identical to nucleotides 1-45 ofSEQ ID NO: 8.

C67. The method according to C65, wherein the codons of the N-terminaltail of the ORF2086 variant are 100% identical to nucleotides 4-45 ofSEQ ID NO: 8.

C68. The method according to C65, wherein the codons of the N-terminaltail of the ORF2086 variant are 100% identical to nucleotides 4-42 ofSEQ ID NO: 8.

C69. The method according to C65, wherein the N-terminal tail of theprotein encoded by the ORF2086 variant comprises at least one amino acidsubstitution compared to amino acids 1-15 of SEQ ID NO: 18.

C70. The method according to C65, wherein the N-terminal tail of theprotein encoded by the ORF2086 variant comprises at least one amino acidsubstitution compared to amino acids 2-15 of SEQ ID NO: 18.

C71. The method according to C65, wherein the N-terminal tail of theprotein encoded by the ORF2086 variant comprises two amino acidsubstitutions compared to amino acids 1-15 of SEQ ID NO: 18.

C72. The method according to C65, wherein the N-terminal tail of theprotein encoded by the ORF2086 variant comprises two amino acidsubstitutions compared to amino acids 2-15 of SEQ ID NO: 18.

C73. The method according to any one of C69-72, wherein the amino acidsubstitutions are conservative amino acid substitutions.

C74. The method according to any one of C65-73, wherein the ORF2086variant is an A22 or a B22 variant.

C75. The method according to any one of C55-74 wherein expression isinduced by addition of IPTG.

C76. The method according to any one of C55-75, wherein the bacteria isE. coli.

What is claimed is:
 1. An immunogenic composition comprising animmunologically effective amount of a first non-pyruvylatednon-lipidated polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 19, wherein the first non-pyruvylated non-lipidatedpolypeptide lacks cysteine at the N-terminus of SEQ ID NO: 19 and doesnot exhibit a mass shift of +70 Da compared to the correspondingwad-type non-lipidated polypeptide as measured by mass spectrometry. 2.The immunogenic composition of claim 1, wherein the firstnon-pyruvylated non-lipidated polypeptide is encoded by a nucleotidesequence comprising SEQ ID NO:
 52. 3. The immunogenic composition ofclaim 1, further comprising an immunologically effective amount of animmunologically effective amount of a non-lipidated N. meningitidis,serogroup B, 2086 Subfamily A polypeptide.
 4. The immunogeniccomposition of claim 6, wherein the Subfamily A polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO:
 55. 5. The immunogeniccomposition of claim 1, further comprising an immunologically effectiveamount of a second non-pyruvylated non-lipidated polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 44, wherein the secondnon-pyruvylated non-lipidated polypeptide lacks cysteine at theN-terminus of SEQ ID NO: 44 and does not exhibit a mass shift of +70 Dacompared to the corresponding wild-type non-lipidated polypeptide asmeasured by mass spectrometry.
 6. The immunogenic composition of claim1, further comprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 57, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:57 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 7. The immunogenic composition of claim 1, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 58, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:58 and does not exhibit a mass shift of +70 Da compared to the,corresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 8. The immunogenic composition of claim 1, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 59, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:59 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 9. The immunogenic composition of claim 1, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 60, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:60 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 10. The immunogenic composition of claim 1, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 62, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:62 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 11. The immunogenic composition of claim 1, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 64, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:64 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 12. An immunogenic composition comprising animmunologically effective amount of a non-pyruvylated non-lipidatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:62, wherein the non-pyruvylated non-lipidated polypeptide lacks cysteineat the N-terminus of SEQ ID NO: 62 and does not exhibit a mass shift of+70 Da compared to the corresponding vi ld-type non-lipidatedpolypeptide as measured by mass spectrometry.
 13. The immunogeniccomposition of claim 12, wherein the non-pyruvylated non-lipidatedpolypeptide is encoded by a nucleotide sequence comprising SEQ ID NO:61.
 14. The immunogenic composition of claim 12, further comprising animmunologically effective amount of an immunologically effective amountof a non-lipidated N. meningitidis, serogroup B, 2086 Subfamily Apolypeptide.
 15. The immunogenic composition of claim 14, wherein theSubfamily A polypeptide comprises the amino acid sequence set forth inSEQ ID NO:
 55. 16. The immunogenic composition of claim 12, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 44, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:44 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 17. The immunogenic composition of claim 12, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 57, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:57 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 18. The immunogenic composition of claim 12, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 58, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:58 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 19. The immunogenic composition of claim 12, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 59, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:59 and does not exhibit a mass shift of +70 Da compared to the,corresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 20. The immunogenic composition of claim 12, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 60, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:60 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.
 21. The immunogenic composition of claim 12, furthercomprising an immunologically effective amount of a secondnon-pyruvylated non-lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 64, wherein the second non-pyruvylatednon-lipidated polypeptide lacks cysteine at the N-terminus of SEQ ID NO:64 and does not exhibit a mass shift of +70 Da compared to thecorresponding wild-type non-lipidated polypeptide as measured by massspectrometry.